ABSTRACT The distal region of the uterine (Fallopian) tube is commonly associated with high-grade serous carcinoma (HGSC), the predominant and most aggressive form of ovarian or

extra-uterine cancer. Specific cell states and lineage dynamics of the adult tubal epithelium (TE) remain insufficiently understood, hindering efforts to determine the cell of origin for

HGSC. Here, we report a comprehensive census of cell types and states of the mouse uterine tube. We show that distal TE cells expressing the stem/progenitor cell marker _Slc1a3_ can

differentiate into both secretory (_Ovgp1_+) and ciliated (_Fam183b_+) cells. Inactivation of _Trp53_ and _Rb1_, whose pathways are commonly altered in HGSC, leads to elimination of targeted

therapeutic targets. SIMILAR CONTENT BEING VIEWED BY OTHERS REDUCED _SKP1_ AND _CUL1_ EXPRESSION UNDERLIES INCREASES IN CYCLIN E1 AND CHROMOSOME INSTABILITY IN CELLULAR PRECURSORS OF

HIGH-GRADE SEROUS OVARIAN CANCER Article Open access 17 March 2021 SILENCING PTEN IN THE FALLOPIAN TUBE PROMOTES ENRICHMENT OF CANCER STEM CELL-LIKE FUNCTION THROUGH LOSS OF PAX2 Article

Open access 07 April 2021 L1CAM IS REQUIRED FOR EARLY DISSEMINATION OF FALLOPIAN TUBE CARCINOMA PRECURSORS TO THE OVARY Article Open access 12 December 2022 INTRODUCTION Ovarian cancer is

the sixth leading cause of death for women in the United States1. High-grade serous carcinoma (HGSC) is the most common and aggressive type of ovarian cancer2,3. Over 80% of HGSC are

detected at advanced stage and have limited treatment options2,4,5. This is attributed to latent progression of the disease with lack of early symptoms and detection methods2. Detection and

treatment of HGSC at earlier stages could be crucial to improving the prognosis of patients with this malignancy. However, identification of new diagnostic markers and therapeutic targets is

hindered by our inadequate knowledge about the cells in which HGSC originates and the mechanisms underlying disease initiation. The location of HGSC initiation has long been debated, but

the emerging consensus is that both the ovarian surface epithelium (OSE) and the tubal epithelium (TE) of the uterine tube, also known as the oviduct or Fallopian tube, have potential to

progress into HGSC6,7,8,9,10,11. While cancer-prone stem/progenitor cells have been described for the OSE12,13, the cell of origin of HGSC arising from TE remains unclear. It has been shown

that the majority of familial HGSC cases may begin with the appearance of early dysplastic lesions, TP53 signatures, and serous tubal intraepithelial carcinomas (STICs)6,14. These lesions

are found exclusively in the distal region of the uterine tube2,14,15. Both TP53 signatures and STICs lack ciliation, express the transcriptional factor PAX8, and harbor mutations in the

_TP53_ gene (also known as _Trp53_ in the mouse), which encodes for p53. _TP53_ mutations are the most frequent genetic alterations in HGSC, being present in over 96% of cases16,17.

Consistent with these observations, early dysplastic lesions can be induced by inactivation of tumor suppressor genes commonly associated with human HGSC, such as _Trp53_, _Brca1_, _Brca2,

Pten_, and _Rb1_ in _Pax8_-expressing tubal epithelial cells of the mouse uterine tube18,19. In both humans and rodents, uterine tubes consist of distal (infundibulum, ampulla, and

ampullary-isthmic junction) and proximal regions (isthmus and intramuscular utero-tubal junction). The uterine tube is formed by the simple pseudostratified TE surrounded by a thin stromal

layer, two circular smooth muscle layers, and the mesothelium. Two main cellular components of the TE are ciliated cells (also known as multi-ciliated cells), predominantly located in the

distal regions of the tube, and secretory cells, which are more abundant in the proximal regions. Additionally, there are basal cells, representing intraepithelial T-lymphocytes and peg

cells. Peg cells have been described as either exhausted secretory cells or CD44+ progenitor cells20,21,22. Previous mouse lineage-tracing studies have reported that cells expressing _Pax8_

have the capacity to self-renew and differentiate into ciliated cells in both distal and proximal regions after labeling during embryonic or prepubertal development23. However, recent

studies based on immunophenotyping, lineage tracing, and limited single cell RNA-sequencing (scRNA-seq) suggest presence of distinct cell lineages in the distal (_Pax8_+) and the proximal

(_Pax2_+) regions of the adult mouse uterine tube24. It is well established that many types of cancer arise from stem cell niches25,26,27. Previously, using a genetically defined mouse

model, we have shown that OSE stem/progenitor cells can be efficiently transformed after inactivation of tumor suppressor genes _Trp53_ and _Rb1_ and lead to HGSC formation12,13,28. Notably,

tumors arising from non-stem OSE cells were slow-growing and non-metastatic. However, in other cancer types, neoplasms may originate from differentiated or transitional state cells that

have acquired some stem cell properties28,29,30. It has been hypothesized that some ovarian carcinomas may arise from the ciliated cell lineage31. However, no direct experimental data has

been offered to support this idea. Here, we conduct scRNA-seq to establish a comprehensive census of cell types found in the mouse uterine tube. Proximal and distal sections are sequenced

separately to investigate characteristics underscoring the distal region’s predisposition towards cancer initiation. By using a combination of computational lineage trajectory projections

and genetic cell fate, we identify a TE stem/progenitor cell population and interrogate unique epithelial cell states for their propensity for malignant transformation. These studies reveal

that pre-ciliated cells may serve as a specific cell state susceptible to malignant transformation. RESULTS CENSUS OF CELL TYPES OF THE MOUSE UTERINE TUBE The mouse uterine tube is divided

along the distal and proximal axis (Fig. 1a). We collected 62 uterine tubes from 31 adult mice, separated distal and proximal regions, and processed them for scRNA-seq (Supplementary Figs. 

1, 2) by region. After sequencing and data preprocessing of distal region samples, 16,583 high-quality cells remained. Following Harmony integration32, we identified 18 clusters by Louvain

clustering contributing to epithelial, stromal, and immune compartments, which we visualized using the uniform manifold approximation and projection (UMAP) (Fig. 1b). Features defining each

cluster were found using general markers known to define each cell type (Fig. 1c). Among the fibroblast populations were three clusters noted as Fibroblast 1 (_Ramp3_+, _Lsamp_+, _Htra3_+),

Fibroblast 2 (_Plac9a_+, _Mgp_+, _Gpx3_+), and Fibroblast 3 (_Nbl1_+, _Cacna1d_+, _Sfrp2_+). Epithelial cells were characterized by epithelial markers (_Epcam_ and _Krt8_), and further

epithelial specificity was achieved with secretory (_Ovgp1_) and ciliated (_Foxj1_) markers. Stem-like epithelial clusters were identified separately as Stem-like Epithelial 1 (_Slc1a3_+,

_Itga6_+, _Lamc2_+) and Stem-like Epithelial 2 (_Cebpd_+, _Hbegf_+, _Kctd14_+). Additional cell types were noted within our dataset to be mesothelial and luteal cells. CHARACTERIZATION OF

DISTAL EPITHELIAL CELL STATES To further investigate the unique cell states of the TE, we subset the epithelial clusters of the distal uterine tube cell types. We specifically subset from

the secretory, ciliated, and stem-like epithelial cell clusters (Supplementary Fig. 1). After processing the distal epithelial subset, we identified 9 clusters consisting of ciliated,

secretory, stem-like, and transitional cell states (Fig. 2a). We further employed Potential of heat diffusion for affinity-based transition embedding (PHATE)33 to better visualize how tubal

epithelial cells progress in a two-dimensional representation similar to UMAP (Fig. 2b). General epithelial markers (_Epcam_, _Krt8_), secretory markers (_Ovgp1_, _Sox17_), and ciliated

markers (_Fam183b, Foxj1_) were used to classify groups of epithelial cells that are known to reside in the TE. Additional features were found using the Wilcoxon Rank Sum Test to determine

cluster-specific markers (Fig. 2c). One cluster within the epithelial subset shared features of fibroblasts, which indicated that this population was most likely a doublet (Supplementary

Fig. 3). In agreement with previous studies21,34, ciliated cells and their precursors were mainly located in the distal region (32% distal vs 10% proximal), while secretory cells

predominantly populated the proximal region (30% distal vs 65% proximal; Supplementary Table 1). Characterization of the 1,785 high quality proximal cells and the 528 cells within the

proximal epithelial subset led to the identification of 6 clusters mainly consisting of secretory cells (Supplementary Fig. 4). Comparison of the clusters identified in distal and proximal

regions only showed strong correlation between a subset of secretory cell states (Supplementary Fig. 5). General expression of stem-like markers indicated that _Slc1a3_ might serve as a

potential marker for epithelial stem/progenitor cells that give rise to both secretory and ciliated cells (Fig. 2c and Supplementary Fig. 6). _Slc1a3_ was previously described as a marker of

a cell population containing distinct skin epithelial stem cells35,36. A pseudotime trajectory was calculated with Monocle337 and overlayed on the PHATE embedding (Fig. 2d). In the

resulting PHATE representation, _Slc1a3_ expression is located at the center with secretory and ciliated branches split by the suspected stem/progenitor cell state (Fig. 2e). In this

representation, _Pax8_ expression is present in _Slc1a3_+ cells and extends towards early cilia-forming cells (Fig. 2f). In the distal region _Pax8_ expression was mainly detected in

stem/progenitor cell cluster (81% of cells) and transitional pre-ciliogenic state cells (49%). Only 3% of ciliated cells and 28% of secretory cells were _Pax8_+ (Fig. 2c, and Supplementary

Table 1). In the proximal region 67% of putative stem/progenitor cells expressed _Pax8_, while both secretory and ciliated clusters contained about 29% of _Pax8+_ cells each (Supplementary

Fig. 4 and Supplementary Table 1). Although the capture of _Pax8_ transcripts may be limited due to dropouts from scRNA-seq, presence of PAX8 expression in SLC1A3+ cells, and its lack in

some secretory cells was confirmed by immunostaining (Supplementary Figs. 7 and 8). _SLC1A3_+ EPITHELIAL CELLS ARE STEM/PROGENITOR CELLS FOR THE TE To test if _Slc1a3_+ epithelial cells are

stem/progenitor cells of the TE, cell lineage-tracing and organoid studies were conducted in Slc1a3-CreERT Ai9 mice. In these mice, administration of tamoxifen allows for Cre-_loxP_ mediated

induction of tdTomato expression in _Slc1a3_+ cells (Fig. 3a). One day after tamoxifen induction tdTomato expression was mainly detected in the distal TE (Fig. 3b, c). Within 30 days after

tamoxifen injection, tdTomato+ cells expanded to form clusters and persisted for over 1 year (Fig. 3b–f). A fraction of tdTomato cells continued to express SLC1A3 indicative of their

long-living potential. Single low dose treatments of tamoxifen yielded corroborating results, in which single cells formed clusters of tdTomato+ cells 30 days after injection (Supplementary

Fig. 9). In agreement with recent studies showing no significant impact of estrous cycle on gene expression in the TE38, tamoxifen administration showed no significant impacts on the

presence of SLC1A3+ cells 1 and 30 days after injection (Supplementary Table 2). SLC1A3+ STEM/PROGENITOR CELLS FORM CILIATED ORGANOIDS WITH HIGH EFFICIENCY Consistent with previous studies

of mouse and human TE20,39,40, organoids were formed by harvesting the mouse distal TE. The organoid-forming ability of tdTomato+ cells from Slc1a3-CreERT Ai9 mice was confirmed with

isolation from the distal region after separation by fluorescence-activated cell sorting (FACS) (Supplementary Fig. 10a–d). To further distinguish the SLC1A3+ stem/progenitor cells from

others within the TE, we completed magnetic-activated cell sorting (MACS) to split pure epithelial cell pools into SLC1A3+ and SLC1A3- groups (Supplementary Fig. 10e and Supplementary Table 

3). Consistent with previous FACS experiments, organoids derived from SLC1A3+ epithelial cells formed organoids at a significantly higher rate as compared to organoids formed by SLC1A3-

cells (Fig. 4a). After 14 days of culture, organoids grew to similar sizes (Supplementary Fig. 10f, g) and contained OVGP1+ cells in both groups (Fig. 4b). However, organoids formed from

SLC1A3+ cells were marked by a significantly larger population of ciliated cells (Fig. 4c–e), thereby supporting bidirectional differentiation potential of SLC1A3+ cells. In summary, based

on general expression of stem-like markers, computational lineage trajectory projections, cell lineage tracing, and organoid assays, _Slc1a3_+ epithelial cells represent stem/progenitor

cells for the distal TE and contribute to the long-term maintenance of both ciliated and secretory epithelial cells within the uterine tube. _SLC1A3_+ EPITHELIAL CELLS ARE NOT CANCER-PRONE

To investigate the role of _Slc1a3_+ cells in malignant transformation, we have prepared Slc1a3-CreERT _Trp53__loxP/loxP_ _Rb1__loxP/loxP_ Ai9 mice. However, no TE neoplastic lesions were

observed in these mice (_n_ = 19) by 1 year after tamoxifen administration (Fig. 5a). Large clusters of TE cells expressed tdTomato at the time of collection. Cre-mediated excision of _Rb1_

but not _Trp53_ was detected by microdissection-PCR assay in all tested samples (Fig. 5b). Evaluation of the TE showed increased apoptotic rate 1 day after tamoxifen administration in mice

with either _Trp53_/_Rb1_ or _Trp53_ mutations. (Fig. 5c, d). Thus, _Trp53_ inactivation leads to elimination of targeted _Slc1a3_+ cells by apoptosis, thereby explaining their resistance to

malignant transformation. PRE-CILIATED CELLS ARE SUSCEPTIBLE TO MALIGNANT TRANSFORMATION Consistent with previous reports18,19, we have observed that the TE can be transformed by

conditional inactivation of _Trp53_ and _Rb1_ in cells expressing _Pax8_ (Fig. 6a, b, Supplementary Table 4). Early dysplastic lesions (6 out of 12 cases), and/or HGSC (3 out of 12 cases)

were detected in 58% of mice by 400 days post induction (DPI). These data suggest that a subset of _Pax8_+ cells is susceptible to malignant transformations, distinct from _Slc1a3_+

stem/progenitor cells that do not give rise to malignancies. To define a unique population of _Pax8_-expressing cells implicated in malignant transformation, we leveraged our scRNA-seq

analysis to identify a group of cells expressing _Pax8_ but not _Slc1a3_ in the distal region of the uterine tube. Strikingly, the only cell state exhibiting _Pax8_ expression without

concomitant _Slc1a3_ expression displayed features consistent with pre-ciliated cells (Fig. 6c–e). We subset the branched trajectory of secretory and ciliated cells from _Slc1a3_+

progenitors and formed 20 pseudotime bins equally divided along the trajectory to total about 150 cells per bin (Fig. 6c, d). We have found that _Pax8_+, _Slc1a3_- cells are present as a

transitional state along the ciliogenic lineage. Such cells are characterized by expression of _Krt5_, _Prom1_, and _Trp73_ (Fig. 6d–f). Notably, expression of these genes negatively

correlates with _Trp53_ expression, thereby suggesting reduced requirement for _Trp53_ during ciliogenesis (Fig. 6d and Supplementary Fig. 11). At the same time, the majority of other

putative driver genes identified in human HGSC13,41, including _Rb1_, _Nf1_, and _Pten_, are preferentially expressed in stem/progenitor populations along the pre-ciliogenesis trajectory.

Expression of _Brca1, Brca2_ and _Csmd3_ is mainly present in cilia-forming cells (Supplementary Fig. 11). To characterize _Krt5_+ pre-ciliated cells, we prepared Krt5-CreERT Ai9 mice. In

agreement with our single cell transcriptome findings, _Krt5_+ cells represented a small fraction (0.83%) of pre-ciliated cells (FOXJ1±) of the distal TE (Fig. 7a, b, Supplementary Table 4).

Consistent with transitional state of _Krt5_+ cells, their progeny mainly presented with ciliated marker expression (FOXJ1 and TRP73) 30 days after tamoxifen treatment (Fig. 7a, b). _Krt5_+

cells of the TE increased in their number as typical for transit-amplifying cells (Supplementary Table 5). Few _Krt5_+ cells were also detected in the OSE. However, these cells decreased in

number by 30 DPI (Supplementary Table 5). To test if pre-ciliated cells are susceptible to malignant transformation, we prepared Krt5-CreERT _Trp53__loxP/loxP_ _Rb1__loxP/loxP_ Ai9 mice. By

200 DPI 16 out of 21 mice (76%) developed neoplastic lesions (Fig. 7c–f, Supplementary Fig. 12 and Supplementary Table 4). Early dysplastic lesions were found in 12 out of 21 mice (57%).

Such lesions showed a range of features from mild cellular atypia (Fig. 7c and Supplementary Fig. 12a, n = 5) to more pronounced cellular atypia, loss of cell polarity, and high

proliferative index typical for STICs (Fig. 7d, _n_ = 8). All dysplastic lesions presented with PAX8 expression and lack of ciliation. They were also marked by elevated expression of HGSC

markers, P16, and Wilms tumor 1 (WT1, Fig. 7c). Notably, 9 out of 12 early lesions (75%) were located near TE-mesothelial junctions (Fig. 7c and Supplementary Fig. 12a), similarly to the

previously described location of human STICs in close vicinity to the tubal-peritoneal junction14,15. Four mice (19%) presented with carcinomas similar to human HGSC, according to their

slit-like and solid patterns of growth, expression of PAX8, WT1, P16 and high levels of proliferation and stromal invasion (Fig. 7e). Three of such tumors spread peritoneally (Fig. 7f) and

invaded ovarian fat pad (Supplementary Fig. 12b). No early dysplastic lesions or overt carcinomas were detected in the OSE. The earliest atypical lesions were observed at 104 DPI as compared

to 154 DPI in experiments with inactivation of _Trp53_ and _Rb1_ in _Pax_8+ cells. According to tdTomato cell labeling at 1 DPI, the pool of targeted _Krt5_+ cells (0.83%) was significantly

smaller than that of _Pax8_+ cells (19%, _P_ = 0.0007; Supplementary Table 4). These observations support the notion that cells in a transitional state along ciliogenesis are cancer-prone.

DISCUSSION Our single-cell transcriptomics allowed for unique cell states and cell lineage hierarchy to be identified in the context of healthy adult mouse uterine tubes (Fig. 8). Further

experimentation has confirmed that _Slc1a3_+ cells are long living and give rise to both secretory (OVGP1+) and ciliated (FAM183B+) cells in the distal region of the uterine tube. Notably,

expression of _Pax8_ was mainly detected in stem/progenitor cells and transitional cell states. Our findings confirm previous observations that _Pax8_+ cells can differentiate into ciliated

cells23. However, they also indicate that mature secretory cells do not give rise to ciliated cells. We could not observe any significant contribution of _Slc1a3_+ cells to cells of the

proximal uterine tube. This can be explained by the existence of developmentally distinct cell lineages forming the proximal region24. Our current study shows that unlike stem/progenitor

cells of the OSE, _Slc1a3_+ TE stem/progenitor cells do not undergo malignant transformation after Cre-mediated inactivation of _Trp53_ and _Rb1_. This was not due to the lack of Cre

activity in _Slc1a3+_ cells. Long-term contribution of tdTomato+ cells to the TE was observed in all uterine tubes by 360 days after induction of Cre expression. Furthermore, all samples

contained an excised _Rb1_ allele. However, we observed only intact floxed _Trp53_ in tdTomato+ TE cells and increased apoptotic index 1 day after induction of Cre expression. Deletion of

_Trp53_ alone was sufficient to induce apoptosis, thereby supporting the notion that inactivation of _Trp53_ is incompatible with long-term survival of _Slc1a3+_ cells. By comparative

evaluation of _Pax8+_ cells lacking concomitant _Slc1a3_ expression, we identified a cancer-prone cell population along the ciliogenic lineage. This observation is consistent with prevalence

of early dysplastic lesions, TP53 signatures and STICs in the distal, ciliated cell-rich, region of the uterine tube2,14,15. Furthermore, the majority of HGSC putative driver genes13,41,

either preferentially expressed in stem/progenitor cells with a bias towards pre-ciliogenic trajectory (_Rb1, Nf1_ and _Pten)_, or mainly detected in cilia-forming cells (_Brca1_, _Brca2_

and _Csmd3_). Thus, aberrations in these genes may have the most transformation potential in the context of pre-ciliogenic cell state, as opposed to secretory differentiation. According to

our analysis of branched trajectory of secretory and ciliated cells from _Slc1a3_+ progenitors and cell fate experimental tracing, _Krt5+_ cells are in a transitional pre-ciliated cell

state. Such cells also express _Prom1_ and _Trp73_. Consistent with our findings, a previous lineage tracing study suggested that _Prom1_+ cells represent a state transitional to

ciliation42. Our previous studies have shown that ciliated _Foxj1_+ cells are not transformed by inactivation of _Trp53_ and _Rb1_19. Hence cancer-susceptibility of TE cells deficient for

these genes is limited to a transitional pre-ciliated cell state marked by expression of _Krt5, Prom1_ and _Trp73_. _KRT5, PROM1_ and _TP53_ have been linked to human ovarian cancer.

Expression of both _KRT5_ and _PROM1_ are associated with HGSC progression43,44. _TP73_, encoding human p73, is upregulated in many cases of epithelial ovarian cancers and modulates the

sensitivity of ovarian cancers to chemotherapy (reviewed in ref. 31). Notably, _Trp73_ is a critical regulator of ciliogenesis and its inactivation results in ciliopathies45,46,47. Among

downstream targets of _Trp73_, are micro-RNAs of the miR-34 family48. These micro-RNAs are also an essential component for ciliogenesis49. At the same time, inactivation of _mir-34_ family

of genes is commonly observed in HGSC50, suggesting a link between regulation of ciliogenesis and ovarian cancer. p73 encoded by _TP73/Trp73_ is a homolog of the tumor suppressor

transcriptional factor p53. Some functions of p53 and p73 are redundant, as evident by the ability of p73 to activate p53-regulated genes in growth suppression and apoptosis induction.

However, other functions do remain unique to either p53 or p73, as evidenced by the lack of ciliation abnormalities in mice null for _Trp53_ and rare mutations of _TP73_ in cancers51.

Consistent with distinct functions of p53 and p73 in TE cells, we have observed that _Trp73_ expression negatively correlates with _Trp53_ expression. Our studies show that _Slc1a3+_

stem/progenitor cells express _Trp53_ and its inactivation is incompatible with their survival. Unlike _Slc1a3_+ cells, transitional pre-ciliated cells express both _Trp53_ and _Trp73_. This

suggests that _Trp73_ expression protects cells from apoptosis but not from genomic instability triggered by _Trp53_, thereby leading to the preferential transformation of pre-ciliated

cells. Further studies are required to understand how transition from p53-regulated programs in stem/progenitor cells to p73-controlled ciliogenesis programs may predispose to cancer. We

have observed preferential location of early dysplastic lesions near the junction between TE and mesothelium, in close similarity to their human counterparts14,15. Junction areas (aka

epithelial transitional zones) are anatomically defined regions of organs where two different types of epithelial tissue meet. Such junction areas are known to be predisposed to cancer in

many locations such as the ovarian hilum region, the gastric squamo-columnar junction, the corneal limbus region, the anal canal, and the uterine cervix. They also contain stem cell niches

(reviewed in ref. 26). The existence of a cancer-prone cells in junction sites has been demonstrated definitively in the ovarian hilum region and the gastric squamo-columnar

junction12,13,28. Our current observation reinforces the notion that high cancer frequency at the junction areas can be explained by the presence of cancer-prone stem cell niches therein.

Further studies should specifically evaluate the mechanisms facilitating preferential transformation of pre-ciliogenic cells at the tubal-mesothelial junction. There are potential

limitations within this study. The use of scRNA-seq may be limited by its low capture efficiency and high level of noise. While scRNA-seq may fail to capture all transcripts in each cell,

the technique was sufficient for identifying markers of cell states within the distal TE. Furthermore, organoids were generated from pools of epithelial cells split by their SLC1A3

expression. However, there may be other epithelial cell states that are less likely to form organoids among the SLC1A3+ and SLC1A3- populations, which may impact the organoid formation rate.

Finally, we explore cancer-prone cell states by inactivating _Trp53_ and _Rb1_. Inactivation of these genes leads to HGSC arising from pre-ciliated TE cells. However, other combinations of

driver mutations may transform other cell states and/or produce different types of ovarian carcinoma. In summary, our study establishes the cell hierarchy, cell lineage dynamics, and

identity of stem/progenitor cells in the distal TE. Furthermore, we show resistance of TE stem/progenitor cells to malignant transformation and provide direct experimental evidence for

cancer propensity of cells in the transitional pre-ciliated state. These findings explain the preferential appearance of neoplastic lesions in the distal region of the uterine tube and point

to the pre-ciliation state as a diagnostic and therapeutic target. METHODS EXPERIMENTAL ANIMALS The Tg(Slc1a3-cre/ERT)1Nat/J (Slc1a3-CreERT, Stock number 012586), Tg(Pax8-rtTA2S*M2)1Koes/J

(Pax8-rtTA, stock number 007176_)_, Tg(tetO-Cre)1Jaw/J (Tre-Cre, stock number 006234), Tg(KRT5-cre/ERT2)2Ipc/Jeldj (K5-Cre-ERT2, Stock number 018394), _Gt(ROSA)26Sor__tm9(CAG-tdTomato)Hze_

(_Rosa-loxp-stop-loxp-_tdTomato/Ai9, Stock number 007909), and C57BL6 (Stock number 000664) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The _Trp53__loxP/loxP_ and

_Rb1__loxP/loxP_ mice, which have _Trp53_ and _Rb1_ genes flanked by _loxP_ alleles, respectively, were a gift from Dr. Anton Berns. Mice were bred on a mixed FVB/N and BL6/J background.

Their genotype was confirmed by PCR amplification (Supplementary Table 6). Only females were used for our studies of the female reproductive tract. The number of animals used in every

experiment is indicated as biological replicates in figure legends and supplementary tables. Animals were euthanized if becoming moribund or developing tumors over 1 cm in diameter. Maximal

allowed tumor size/burden was not exceeded. All the experiments and maintenance of the mice followed ethical regulations for animal testing and research. They were approved by the Cornell

University Institutional Laboratory Animal Use and Care Committee (protocols numbers 2000-0116 and 2001-0072). Mice were housed within a 10/14 light cycle. The lights came on at 5 a.m. and

went off at 7 p.m., the humidity ranged from 30–70 %, and the ambient temperature was kept at 72 °F ± 2 °F. DOXYCYCLINE AND TAMOXIFEN INDUCTION For estimation of target cell frequency 6–8

week-old Pax8-rtTA Tre-Cre Ai9 mice received a single dose (12 μl g−1 body weight) of doxycycline (6.7 mg ml−1 in sterile PBS) by intraperitoneal (i.p.) injection. Identical injection

schedule was used for tumor induction experiments with 6–10 week-old Pax8-rtTA Tre-Cre _Trp53__loxP/loxP_ _Rb1__loxP/loxP_ Ai9 mice and control mice. In our cohort, about 10% of TRE-Cre

_Trp53__loxP/loxP_ _Rb1__loxP/loxP_ mice have developed histiocytic sarcomas and have not been included in further analyzes. For tamoxifen induction of Cre expression in cell lineage tracing

experiments 6–8 week-old Slc1a3-CreERT Ai9 mice received 1, 3, and 5 daily i.p. injections of tamoxifen (100 μg/g body weight, 8 μl/g body weight, 12.5 mg/ml in corn oil; Sigma-Aldrich,

T5648). For low dose lineage tracing experiments, mice were treated with a single i.p. injection of tamoxifen at 10 μg/g body weight). In tumor induction experiments, 6–10 week-old mice

Slc1a3-CreERT _Trp53_loxP/loxP _Rb1_loxP/loxP Ai9 mice received i.p. injections every 24 h for 3 days. K5-CreERT2 _Trp53__loxP/loxP_ _Rb1__loxP/loxP_ Ai9 mice of the same age received a

single i.p. injection of tamoxifen. For organoid assays after FACS, 5 Slc1a3-CreERT Ai9 mice 6–12 week-old mice per experiment received a single 36 h tamoxifen pulse (100 μg/g body weight as

described above). All mice were euthanized by CO2 and further analyses were carried out. ANATOMICAL NOMENCLATURE “Uterine tube”, “Fallopian tube”, and “oviduct” are terms commonly used by

different groups of investigators. The term “uterine tube” precisely describes its location and function as a tube leading from the ovary to the uterus. “Fallopian tube,” named after the

16th-century Italian anatomist Gabriele Falloppio, is a more traditional term, but it’s less descriptive anatomically. “Oviduct” is anatomically descriptive, indicating a duct for the ovum

(egg), but it is less specific to human anatomy and is often used in the context of various animals. We use the term “uterine tube” as the most anatomically accurate name for this structure.

The “uterine tube” is also the most unifying term allowing to be efficiently used for comparative human/mouse studies common in experimental research. PATHOLOGICAL EVALUATION All mice in

carcinogenesis experiments were subjected to gross pathology evaluation during necropsy. Particular attention was paid to potential sites of ovarian carcinoma spreading, such as the omentum,

regional lymph nodes, uterus, liver, lung and mesentery. In addition to the ovary, pathologically altered organs, as well as representative specimens of the brain, lung, liver, kidney,

spleen, pancreas and intestine, intra-abdominal lymph nodes, omentum and uterus were fixed in 4% PBS buffered paraformaldehyde. They were then evaluated by microscopic analysis of serial

paraffin sections stained with hematoxylin and eosin and subjected to necessary immunostainings. All early atypical lesions were diagnosed based on their morphology, staining for PAX8,

extent of proliferation and detection of deleted (floxed-out) _Trp53_ and _Rb1_ as described earlier52. STICs were identified based on pronounced cellular atypia, the loss of cell polarity,

high proliferative index, and _Trp53_ deletion. The locations of all lesions were determined by three-dimensional reconstruction of 4-mm-thick serial sections as described previously53.

IMMUNOHISTOCHEMISTRY, APOPTOSIS DETECTION, AND IMAGE ANALYSIS For lineage tracing experiments, Slc1a3-CreERT Ai9 induced mouse tissues were fixed with 4% paraformaldehyde for 1.5 h on ice.

After fixation tissues were washed three times 5 min with PBS at room temperature, embedded in Tissue-Tek O.C.T. compound (ThermoFisherScientific), and frozen on dry ice for 10 min.

Immunofluorescent detection of FAM183B (IF, dilution 1:150) and OVGP1 (IF, dilution 1:600, 1:800, IHC, 1:800, 1:2000–8000) was performed in 7 μm-thick frozen sections according to standard

protocols. For paraffin embedding, tissues were fixed in 4% paraformaldehyde overnight at 4 °C followed by standard tissue processing, paraffin embedding, and preparation of 4 μm-thick

tissue sections. For immunohistochemistry, antigen retrieval was performed by incubation of deparaffinized and rehydrated tissue sections in boiling 10 mM sodium citrate buffer (pH 6.0) for

10 min. The primary antibodies against PAX8 (anti-mouse, IF, dilution 1:100, IHC, 1:5000–10,000, anti-rabbit, IF, 1:400, IHC, 1:4000), and tdTomato/RFP (anti-goat, IF, dilution 1:100,

anti-rabbit, IF, 1:250, IHC, 1:4000) were incubated at 4 °C for overnight, followed by incubation with secondary biotinylated antibodies (dilution 1:200, 45 min, at room temperature, RT).

Modified Elite avidin-biotin peroxidase (ABC) technique (Vector Laboratories, Burlingame, CA, USA; pk-6100) was performed at room temperature for 30 min. Hematoxylin was used as the

counterstain. All primary and secondary antibodies used for immunostaining are listed in Supplementary Table 7. Apoptosis detection was performed on paraffin sections with TUNEL assay

following manufacturer instructions (abcam, ab206386). All stained cells were additionally confirmed by morphological recognition of apoptotic bodies, featuring nuclear fragmentation,

cytoplasmic shrinkage, and blebbing53,54. More than 1200 cells per sample were scored to estimate the apoptotic index. For quantitative studies, sections were scanned with a ScanScope CS2

(Leica Biosystems, Vista, CA), 40x objective, a Zeiss LSM 710 Confocal Microscope for tile scans with a pixel dwell of 1.58 μsec and an averaging of 2 using the ZEN (blue edition, Zeiss)

software, or a Leica TCS SP5 Confocal Microscope, 20x and 40x objective, followed by the analysis with Fiji software (National Institutes of Health, Bethesda, MD, USA). MICRODISSECTION-PCR

Four μm thick paraffin sections were placed on glass slides, stained with H&E, and parallel sections stained for tdTomato were evaluated under microdissection microscope. Cells from HE

stained sections were collected using a 25G1/2 needle, into 0.6 ml Eppendorf tubes filled with lysis buffer, digested in proteinase K, and processed for subsequent PCR

amplification12,52,53,55. TE ORGANOID PREPARATION Mouse tubal epithelial cells (TE) were isolated from Slc1a3-CreERT Ai9 positive and negative littermates. Single cell suspensions for

organoids were adapted from previously described methods7,10,56. Briefly, mouse uterine tubes were collected, washed with wash buffer (Phosphate buffered saline pH 7.4, Thermo Fisher

10010023, 10,000 U/ml Penicillin-Streptomycin, Thermo Fisher 15140122) and digested with digestion buffer (Gibco DMEM/F12, Fisher 11320033; 4 µg/ml Roche Collagenase/Dispase, Sigma

10269638001; 10 µg/ml DNaseI, Sigma 1128459638001) for two rounds (45 min each) at 37 °C. Between rounds, previous digestion buffer was collected and neutralized with 20% FBS containing

media in a new tube. New digestion buffer was added to the tissue and mixed vigorously, and digestion allowed to continue. After neutralization, cells were spun down at 600 × _g_ for 5 min

at 4 °C and mixed with Matrigel. For every 2 mice (4 uterine tubes), 100 µl of Matrigel would be used and plated along the rim of a 24 well tissue culture plate. Plates were then be

incubated in 5% CO2 cell incubator at 37 °C for 60 min, after which Matrigel would be set and organoid media (Gibco Advanced DMEM/F12, Thermo Fisher 12634010; 25% L-WRN Conditioned Medium,

ATCC CRL-3276; 12 mM HEPES, Thermo Fisher 15630080; 1% GlutaMax, Thermo Fisher 35050079; 2% B27, Thermo Fisher 17504044; 1% N2, Thermo Fisher 17502048; 10 ng/ml hEGF, Sigma E9644; 100 ng/ml

Human FGF-10, 1 mM Nicotinamide, Sigma N0636; 10 µM Y-27632 (ROCKi), Millipore 688000; 2.5 µM TGF-B RI Kinase Inhibitor VI, Millipore 616464) was added. For passaging, organoids were

released from Matrigel with organoid harvesting media (R&D 3700-100-01) by pipetting around the rim of the plate. Released cells were transferred to a 15 ml falcon tube and allowed to

chill on ice for 1 h. Cells were then spun down in a refrigerated centrifuge at 600 × _g_ for 5 min at 4 °C. Supernatant was aspirated, and 1 ml of TrypLE (Thermo Fisher 12604013) was added.

Cells were then incubated at 37 °C for 10 min and vigorously pipetted. Cells were then recovered with 20% FBS containing media and spun down at 600xg for 5 min at 4 °C. Supernatant was

aspirated, organoid media was added, and cells were counted. Typically, cells were plated at 500 cells/µl of Matrigel for expansion. To induce Slc1a3-Cre-ERT mediated recombination, 1 µM

4-Hydroxytamoxifen (Selleck Chem S7827) was supplemented to the media for 48 h. FACS PREPARATION AND ANALYSIS Organoid cells were prepared as described above in organoid preparation. After

rescuing cells from digestion, cells were washed in 1x PBS + 1% BSA three times. After the last wash, cells were suspended in 1x PBS + 1% BSA. FACS was performed on a Sony MA900 in two

separate experiments with single replicates. Data was acquired using Sony MA-900 complementary software. Analysis was performed using FlowJo software (BD Biosciences). Post FACS, organoids

were visualized under a microscope to check for tdTomato expression. Slc1a3-CreERT negative littermates were used to gate negative controls (Supplementary Fig. 13). After gating single

cells, Sytox Blue was used to gate for live or dead populations (Supplementary Fig. 13c). From the live cell population, tdTomato+ cells were determined by using Slc1a3-CreERT negative

litter mates (Supplementary Fig. 13d). Cells were then plated in Matrigel at a density of 1000 cells per 10 μl. 1000 SLC1A3+ cells and 30,000 SLC1A3- cells were plated. Assessment of

organoid forming potential was calculated by counting the number of organoids formed against the number of cells sorted. Each replicate contained uterine tubes pooled from at least 5 mice.

MACS PREPARATION AND ANALYSIS Single cell suspensions from the distal mouse uterine tube were prepared as described above from 10 C57BL6 adult female mice aged 6–12 weeks. Magnetic activated

cell sorting was performed following the manufacturer protocol (Invitrogen 11533D). Negative selection was first performed to remove stromal cells by incubating cells with biotinylated

antibodies against CD45 (Biolegend, 103103, 0.5 µg per million cells), TER-119 (Biolegend, 116203, 0.5 µg per million cells), CD140a (Biolegend, 135910, 0.5 µg per million cells), CD31

(Biolegend, 102503, 0.5 µg per million cells). Positive selection was then performed on the unbound fraction of cells by incubating with a biotinylated antibody against SLC1A3 (Novus

biologicals, NB100-1869B, 0.5 µg per million cells). After removal of the unbound fraction, bound cells were released with 200U of DNase I (Sigma, 1128459638001). Cells were counted and

equal numbers of cells were plated for both the SLC1A3+ and SLC1A3- populations (100 cells/µl Matrigel). 5000 cells were plated per Matrigel droplet in a 24 well plate. Representative images

were taken every few days from plating to day 14. Day 14 organoids were counted for organoid formation rate, organoid size, and sections prepared to assess for expression of organoid

markers. ORGANOID HISTOLOGICAL PREPARATION AND ANALYSIS Organoids were released from Matrigel with Cultrex Organoid Harvesting Solution and chilled on ice for 1 h as per the passaging

protocol. Organoids were then centrifuged in a refrigerated centrifuge at 100 × _g_ for 30 s at 4 °C. Organoids were fixed on ice in 5 ml of 4% PBS-buffered paraformaldehyde while avoiding

exposure to light. Organoids were spun down at 100 × _g_ for 30 s and replaced with 1x PBS to rinse. Organoids were spun down once more at 100 × _g_ for 30 s to aspirate the remaining PBS.

Organoids were then mixed in 200 µl of HistoGel (Epredia, HG4000012) preheated to 65 °C and dispensed into dome-shaped molds for embedding. HistoGel molds set for 10 min prior to standard

tissue processing, paraffin embedding, and preparation of 4-μm-thick tissue sections followed by immunohistochemistry57. SINGLE-CELL RNA-SEQUENCING LIBRARY PREPARATION For TE single cell

expression and transcriptome analysis we isolated TE from C57BL6 adult female mice at the estrus stage of estrous cycle. In 3 independent experiments a total of 62 uterine tubes were

collected. Each uterine tube was placed in sterile PBS containing 100 IU ml−1 of penicillin and 100 µg ml−1 streptomycin (Corning, 30-002-Cl), and separated in distal and proximal regions.

Tissues from the same region were combined in a 40 µl drop of the same PBS solution, cut open lengthwise, and minced into 1.5–2.5 mm pieces with 25G needles. Minced tissues were transferred

with help of a sterile wide bore 200 µl pipette tip into a 1.8 ml cryo vial containing 1.2 ml A-mTE-D1 (300 IU ml−1 collagenase IV mixed with 100 IU ml−1 hyaluronidase; Stem Cell

Technologies, 07912, in DMEM Ham’s F12, Hyclone, SH30023.FS). Tissues were incubated with loose cap for 1 h at 37 °C in a 5% CO2 incubator. During the incubation tubes were taken out 4

times, and tissues suspended with a wide bore 200 µl pipette tip. At the end of incubation, the tissue-cell suspension from each tube was transferred into 1 ml TrypLE (Invitrogen, 12604013)

pre-warmed to 37 °C, suspended 70 times with a 1000 µl pipette tip, 5 ml A-SM [DMEM Ham’s F12 containing 2% fetal bovine serum (FBS)] were added to the mix, and TE cells were pelleted by

centrifugation 300 × _g_ for 10 min at 25 °C. Pellets were then suspended with 1 ml pre-warmed to 37 °C A-mTE-D2 (7 mg ml−1 Dispase II, Worthington NPRO2, and 10 µg ml−1 Deoxyribonuclease I,

Stem Cell Technologies, 07900), and mixed 70 times with a 1000 µl pipette tip. 5 ml A-mTE-D2 was added and samples were passed through a 40 µm cell strainer, and pelleted by centrifugation

at 300 × _g_ for 7 min at +4 °C. Pellets were suspended in 100 µl microbeads per 107 total cells, or fewer, and dead cells were removed with the Dead Cell Removal Kit (Miltenyi Biotec,

130-090-101) according to the manufacturer’s protocol. Pelleted live cell fractions were collected in 1.5 ml low binding centrifuge tubes, kept on ice, and suspended in ice cold 50 µl

A-Ri-Buffer (5% FBS, 1% GlutaMAX-I, Invitrogen, 35050-079, 9 µM Y-27632, Millipore, 688000, and 100 IU ml−1 penicillin 100 μg ml−1 streptomycin in DMEM Ham’s F12). Cell aliquots were stained

with trypan blue for live and dead cell calculation. Live cell preparations with a target cell recovery of 5000–6000 were loaded on Chromium controller (10X Genomics, Single Cell 3’ v2

chemistry) to perform single cell partitioning and barcoding using the microfluidic platform device. After preparation of barcoded, next-generation sequencing cDNA libraries samples were

sequenced on Illumina NextSeq500 System. DOWNLOAD AND ALIGNMENT OF SINGLE-CELL RNA SEQUENCING DATA For sequence alignment, a custom reference for mm39 was built using the cellranger (v6.1.2,

10x Genomics) _mkref_ function. The mm39.fa soft-masked assembly sequence and the mm39.ncbiRefSeq.gtf (release 109) genome annotation last updated 2020-10-27 were used to form the custom

reference. The raw sequencing reads were aligned to the custom reference and quantified using the cellranger _count_ function. PREPROCESSING AND BATCH CORRECTION All preprocessing and data

analysis was conducted in R (v.4.1.1 (2021-08-10)). The cellranger count outs were first modified with the _autoEstCont_ and _adjustCounts_ functions from SoupX (v.1.6.1) to output a

corrected matrix with the ambient RNA signal (soup) removed (https://github.com/constantAmateur/SoupX). To preprocess the corrected matrices, the Seurat (v.4.1.1) _NormalizeData_,

_FindVariableFeatures_, _ScaleData_, _RunPCA_, _FindNeighbors_, and _RunUMAP_ functions were used to create a Seurat object for each sample (https://github.com/satijalab/seurat). The number

of principal components used to construct a shared nearest-neighbor graph were chosen to account for 95% of the total variance. To detect possible doublets, we used the package DoubletFinder

(v.2.0.3) with inputs specific to each Seurat object. DoubletFinder creates artificial doublets and calculates the proportion of artificial k nearest neighbors (pANN) for each cell from a

merged dataset of the artificial and actual data. To maximize DoubletFinder’s predictive power, mean-variance normalized bimodality coefficient (BCMVN) was used to determine the optimal pK

value for each dataset. To establish a threshold for pANN values to distinguish between singlets and doublets, the estimated multiplet rates for each sample were calculated by interpolating

between the target cell recovery values according to the 10x Chromium user manual. Homotypic doublets were identified using unannotated Seurat clusters in each dataset with the

_modelHomotypic_ function. After doublets were identified, all distal and proximal samples were merged separately. Cells with greater than 30% mitochondrial genes, cells with fewer than 750

nCount RNA, and cells with fewer than 200 nFeature RNA were removed from the merged datasets. To correct for any batch defects between sample runs, we used the harmony (v.0.1.0) integration

method (https://github.com/immunogenomics/harmony). CLUSTERING PARAMETERS AND ANNOTATIONS After merging the datasets and batch-correction, the dimensions reflecting 95% of the total variance

were input into Seurat’s _FindNeighbors_ function with a k.param of 70. Louvain clustering was then conducted using Seurat’s _FindClusters_ with a resolution of 0.7. The resulting 19

clusters were annotated based on the expression of canonical genes and the results of differential gene expression (Wilcoxon Rank Sum test) analysis. One cluster expressing lymphatic and

epithelial markers was omitted from later analysis as it only contained 2 cells suspected to be doublets. To better understand the epithelial populations, we reclustered 6 epithelial

populations and reapplied harmony batch correction. The clustering parameters from _FindNeighbors_ was a k.param of 50, and a resolution of 0.7 was used for _FindClusters_. The resulting 9

clusters within the epithelial subset were further annotated using differential expression analysis and canonical markers. PSEUDOTIME ANALYSIS PHATE is dimensional reduction method to more

accurately visualize continual progressions found in biological data33. A modified version of Seurat (v4.1.1) was developed to include the “RunPHATE” function for converting a Seurat Object

to a PHATE embedding. This was built on the phateR package (v.1.0.7) (https://github.com/scottgigante/seurat/tree/patch/add-PHATE-again). In addition to PHATE, pseudotime values were

calculated with Monocle3 (v.1.2.7), which computes trajectories with an origin set by the user37,58,59,60. The origin was set to be a progenitor cell state confirmed with lineage tracing

experiments. STATISTICAL ANALYSES Statistical comparisons were performed using a two-tailed unpaired _t_ test and Analysis of Variance (ANOVA) with Tukey-Kramer Multiple Comparisons Test

with InStat 3 and Prism 6 software (GraphPad Software Inc., La Jolla, CA, USA). REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting

Summary linked to this article. DATA AVAILABILITY The raw sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) database under accession code

GSE252786. All processed Seurat objects for scRNA-seq analysis are available in the Dryad repository at https://doi.org/10.5061/dryad.4mw6m90hm [https://doi.org/10.5061/dryad.4mw6m90hm]. All

data generated in this study are provided in the Source Data file. The remaining data are available within the Article, Supplementary or Source Data file. Source data are provided with this

paper. CODE AVAILABILITY All code for data preprocessing and figure generation of scRNA-seq data are available through GitHub (https://github.com/coulterr24/MouseTE_scRNA/) and Zenodo

(https://doi.org/10.5281/zenodo.13830750). CHANGE HISTORY * _ 20 NOVEMBER 2024 A Correction to this paper has been published: https://doi.org/10.1038/s41467-024-54465-x _ REFERENCES *

Siegel, R. L., Giaquinto, A. N. & Jemal, A. Cancer statistics, 2024. _CA Cancer J. Clin._ 74, 12–49 (2024). Article  PubMed  Google Scholar  * Seidman, J. D., Ronnett, B. M., Shih Ie,

M., Cho, K. R. & Kurman, R. J. in _Blaustein’s Pathology of the Female Genital Tract_ (eds Kurman, R. J., Ellenson, L. H. & Ronnett, B. M.) 841–966 (Springer, 2019). * Carlson, J.

W., Gilks, C. B. & Soslow, R. A. _Tumors of the Ovary and Fallopian Tube_. Vol. 16 (American Registry of Pathology, 2023). * Kurman, R. J. & Shih Ie, M. The origin and pathogenesis

of epithelial ovarian cancer: a proposed unifying theory. _Am. J. Surg. Pathol._ 34, 433–443 (2010). Article  PubMed  PubMed Central  Google Scholar  * Landen, C. N. Jr., Birrer, M. J. &

Sood, A. K. Early events in the pathogenesis of epithelial ovarian cancer. _J. Clin. Oncol._ 26, 995–1005 (2008). Article  PubMed  Google Scholar  * Kim, J. et al. Cell origins of

high-grade serous ovarian cancer. _Cancers_ 10, pii: E433 (2018). Article  Google Scholar  * Zhang, S. et al. Both fallopian tube and ovarian surface epithelium are cells-of-origin for

high-grade serous ovarian carcinoma. _Nat. Commun._ 10, 5367 (2019). Article  ADS  PubMed  PubMed Central  Google Scholar  * Lawrenson, K. et al. A study of high-grade serous ovarian cancer

origins implicates the SOX18 transcription factor in tumor development. _Cell Rep._ 29, 3726–3735 e3724 (2019). Article  CAS  PubMed  Google Scholar  * Hao, D. et al. Integrated analysis

reveals tubal- and ovarian-originated serous ovarian cancer and predicts differential therapeutic responses. _Clin. Cancer Res._ 23, 7400–7411 (2017). Article  CAS  PubMed  Google Scholar  *

Lohmussaar, K. et al. Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids. _Nat. Commun._ 11, 2660 (2020). Article  ADS  CAS  PubMed 

PubMed Central  Google Scholar  * Maniati, E. et al. Mouse ovarian cancer models recapitulate the human tumor microenvironment and patient response to treatment. _Cell Rep._ 30, 525–540 e527

(2020). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Flesken-Nikitin, A. et al. Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche.

_Nature_ 495, 241–245 (2013). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Yamulla, R. J., Nalubola, S., Flesken-Nikitin, A., Nikitin, A. Y. & Schimenti, J. C. Most

commonly mutated genes in high-grade serous ovarian carcinoma are nonessential for ovarian surface epithelial stem cell transformation. _Cell Rep._ 32, 108086 (2020). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Schmoeckel, E. et al. LEF1 is preferentially expressed in the tubal-peritoneal junctions and is a reliable marker of tubal intraepithelial lesions. _Mod.

Pathol._ 30, 1241–1250 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Seidman, J. D. Serous tubal intraepithelial carcinoma localizes to the tubal-peritoneal junction: a

pivotal clue to the site of origin of extrauterine high-grade serous carcinoma (ovarian cancer). _Int. J. Gynecol. Pathol._ 34, 112–120 (2015). Article  CAS  PubMed  Google Scholar  * Kuhn,

E. et al. TP53 mutations in serous tubal intraepithelial carcinoma and concurrent pelvic high-grade serous carcinoma-evidence supporting the clonal relationship of the two lesions. _J.

Pathol._ 226, 421–426 (2012). Article  CAS  PubMed  Google Scholar  * Lee, Y. et al. A candidate precursor to serous carcinoma that originates in the distal fallopian tube. _J. Pathol._ 211,

26–35 (2007). Article  CAS  PubMed  Google Scholar  * Perets, R. et al. Transformation of the fallopian tube secretory epithelium leads to high-grade serous ovarian cancer in Brca;Tp53;Pten

models. _Cancer Cell_ 24, 751–765 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fu, D. J. et al. Cells expressing PAX8 are the main source of homeostatic regeneration of

adult endometrial epithelium and give rise to serous endometrial carcinoma. _Dis. Model. Mech._ https://doi.org/10.1242/dmm.047035 (2020). * Paik, D. Y. et al. Stem-like epithelial cells are

concentrated in the distal end of the fallopian tube: a site for injury and serous cancer initiation. _Stem Cells_ 30, 2487–2497 (2012). Article  CAS  PubMed  Google Scholar  * Stewart, C.

A. & Behringer, R. R. Mouse oviduct development. _Results Probl. Cell Differ._ 55, 247–262 (2012). Article  PubMed  Google Scholar  * Aviles, M., Coy, P. & Rizos, D. The oviduct: a

key organ for the success of early reproductive events. _Anim. Front._ 5, 25–31 (2015). Article  Google Scholar  * Ghosh, A., Syed, S. M. & Tanwar, P. S. In vivo genetic cell lineage

tracing reveals that oviductal secretory cells self-renew and give rise to ciliated cells. _Development_ 144, 3031–3041 (2017). CAS  PubMed  Google Scholar  * Ford, M. J. et al. Oviduct

epithelial cells constitute two developmentally distinct lineages that are spatially separated along the distal-proximal axis. _Cell Rep._ 36, 109677 (2021). Article  CAS  PubMed  Google

Scholar  * Flesken-Nikitin, A., Odai-Afotey, A. A. & Nikitin, A. Y. Role of the stem cell niche in the pathogenesis of epithelial ovarian cancers. _Mol. Cell Oncol._ 1,

963431–963437.e963435 (2014). Google Scholar  * Fu, D. J. et al. Stem cell pathology. _Annu. Rev. Pathol._ 13, 71–92 (2018). Article  CAS  PubMed  Google Scholar  * Nassar, D. &

Blanpain, C. Cancer stem cells: basic concepts and therapeutic implications. _Annu. Rev. Pathol._ 11, 47–76 (2016). Article  CAS  PubMed  Google Scholar  * Fu, D. J. et al. Gastric

squamous-columnar junction contains a large pool of cancer-prone immature osteopontin responsive Lgr5(-)CD44(+) cells. _Nat. Commun._ 11, 84 (2020). Article  ADS  CAS  PubMed  PubMed Central

  Google Scholar  * Friedmann-Morvinski, D. et al. Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice. _Science_ 338, 1080–1084 (2012). Article  ADS  CAS 

PubMed  PubMed Central  Google Scholar  * Schwitalla, S. et al. Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties. _Cell_ 152, 25–38

(2013). Article  CAS  PubMed  Google Scholar  * Rohozinski, J., Diaz-Arrastia, C. & Edwards, C. L. Do some epithelial ovarian cancers originate from a fallopian tube ciliate cell

lineage? _Med Hypotheses_ 107, 16–21 (2017). Article  CAS  PubMed  Google Scholar  * Korsunsky, I. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. _Nat.

Methods_ 16, 1289–1296 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Moon, K. R. et al. Visualizing structure and transitions in high-dimensional biological data. _Nat.

Biotechnol._ 37, 1482–1492 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yamanouchi, H., Umezu, T. & Tomooka, Y. Reconstruction of oviduct and demonstration of

epithelial fate determination in mice. _Biol. Reprod._ 82, 528–533 (2010). Article  CAS  PubMed  Google Scholar  * Sada, A. et al. Defining the cellular lineage hierarchy in the

interfollicular epidermis of adult skin. _Nat. Cell Biol._ 18, 619–631 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ghuwalewala, S. et al. Binary organization of epidermal

basal domains highlights robustness to environmental exposure. _EMBO J._ 41, e110488 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cao, J. et al. The single-cell

transcriptional landscape of mammalian organogenesis. _Nature_ 566, 496–502 (2019). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Winkler, I. et al. The cycling and aging

mouse female reproductive tract at single-cell resolution. _Cell_ 187, 981–998 e925 (2024). Article  CAS  PubMed  Google Scholar  * Xie, Y., Park, E. S., Xiang, D. & Li, Z. Long-term

organoid culture reveals enrichment of organoid-forming epithelial cells in the fimbrial portion of mouse fallopian tube. _Stem Cell Res._ 32, 51–60 (2018). Article  CAS  PubMed  Google

Scholar  * Rose, I. M. et al. WNT and inflammatory signaling distinguish human Fallopian tube epithelial cell populations. _Sci. Rep._ 10, 9837 (2020). Article  ADS  CAS  PubMed  PubMed

Central  Google Scholar  * Network, T. C. G. A. R. Integrated genomic analyses of ovarian carcinoma. _Nature_ 474, 609–615 (2011). Article  Google Scholar  * Ford, M. J., Harwalkar, K.,

Kazemdarvish, H., Yamanaka, N. & Yamanaka, Y. CD133/Prom1 marks proximal mouse oviduct epithelial progenitors and adult epithelial cells with a low generative capacity. _Biol. Open_ 12,

https://doi.org/10.1242/bio.059963 (2023). * Kaipio, K. et al. ALDH1A1‐related stemness in high‐grade serous ovarian cancer is a negative prognostic indicator but potentially targetable by

EGFR/mTOR‐PI3K/aurora kinase inhibitors. _J. Pathol._ 250, 159–169 (2020). Article  CAS  PubMed  Google Scholar  * Ricciardelli, C. et al. Keratin 5 overexpression is associated with serous

ovarian cancer recurrence and chemotherapy resistance. _Oncotarget_ 8, 17819–17832 (2017). Article  PubMed  PubMed Central  Google Scholar  * Logotheti, S. et al. Mechanisms of functional

pleiotropy of p73 in cancer and beyond. _Front. Cell Dev. Biol._ 9, 737735 (2021). Article  PubMed  PubMed Central  Google Scholar  * Nemajerova, A. et al. Non-oncogenic roles of TAp73: from

multiciliogenesis to metabolism. _Cell Death Differ._ 25, 144–153 (2018). Article  CAS  PubMed  Google Scholar  * Marshall, C. B. et al. p73 Is required for multiciliogenesis and regulates

the Foxj1-associated gene network. _Cell Rep._ 14, 2289–2300 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Nemajerova, A. et al. TAp73 is a central transcriptional

regulator of airway multiciliogenesis. _Genes Dev._ 30, 1300–1312 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Song, R. et al. miR-34/449 miRNAs are required for motile

ciliogenesis by repressing cp110. _Nature_ 510, 115–120 (2014). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Corney, D. C. et al. Frequent downregulation of miR-34 family in

human ovarian cancers. _Clin. Cancer Res._ 16, 1119–1128 (2010). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Levrero, M. et al. The p53/p63/p73 family of transcription

factors: overlapping and distinct functions. _J. Cell Sci._ 113, 1661–1670 (2000). (PT 10). Article  CAS  PubMed  Google Scholar  * Flesken-Nikitin, A., Choi, K. C., Eng, J. P., Shmidt, E.

N. & Nikitin, A. Y. Induction of carcinogenesis by concurrent inactivation of p53 and Rb1 in the mouse ovarian surface epithelium. _Cancer Res._ 63, 3459–3463 (2003). CAS  PubMed  Google

Scholar  * Nikitin, A. Y. & Lee, W. H. Early loss of the retinoblastoma gene is associated with impaired growth inhibitory innervation during melanotroph carcinogenesis in _Rb_+/- mice.

_Genes Dev._ 10, 1870–1879 (1996). Article  CAS  Google Scholar  * Nikitin, A. Y. et al. Cell lineage-specific effects associated with multiple deficiencies of tumor susceptibility genes in

Msh2(-/-)Rb(+/-) mice. _Cancer Res._ 62, 5134–5138 (2002). CAS  PubMed  Google Scholar  * Matoso, A., Zhou, Z., Hayama, R., Flesken-Nikitin, A. & Nikitin, A. Y. Cell lineage-specific

interactions between Men1 and Rb in neuroendocrine neoplasia. _Carcinogenesis_ 29, 620–628, (2008). Article  CAS  PubMed  Google Scholar  * Ford, M. J., Harwalkar, K. & Yamanaka, Y.

Protocol to generate mouse oviduct epithelial organoids for viral transduction and whole-mount 3D imaging. _STAR Protoc._ 3, 101164 (2022). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Flesken-Nikitin, A., Harlan, B. A. & Nikitin, A. Y. Transplantation into the mouse ovarian fat pad. _J. Vis. Exp._ https://doi.org/10.3791/54444 (2016). * Trapnell, C. et al.

The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. _Nat. Biotechnol._ 32, 381–386 (2014). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Qiu, X. et al. Single-cell mRNA quantification and differential analysis with Census. _Nat. Methods_ 14, 309–315 (2017). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. _Nat. Methods_ 14, 979–982 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references ACKNOWLEDGEMENTS We thank Matalin Pirtz, Nikitin lab for critical reading of the manuscript, Peter A. Schweitzer, Director of the Cornell Genomics Facility for his

invaluable assistance with single-cell RNA sequencing, Tudorita Tumbar, Cornell Molecular Biology, and Genetics, for providing Slc1a3-CreERT Ai9 mice from her long-term cell lineage tracing

experiments and advising on the initial Slc1a3-related experiments, Temirlan Shilikbay, Nazarbayev University, Astana, Kazakhstan for his early contributions to a pilot _Slc1a3_ project, and

Md Mozammal Hossain, Kurtay Ozuner and Derrick Tran for their excellent technical support. This work has been supported by NIH grants (CA182413, CA260115, and CA248524) to A.Y.N., Ovarian

Cancer Research Fund grant (327516) to A.Y.N., Sandra Atlas Bass Endowment for Cancer Research to A.Y.N. and J.C.S., the NSF Graduate Research Fellowship Program (GRFP) awarded to C.Q.R.

(DGE-2139899) and NIH 1S10RR025502 grant to the Cornell Institute of Biotechnology Imaging Facility for the Zeiss LSM 710 Confocal Microscope. AUTHOR INFORMATION Author notes * These authors

contributed equally: Andrea Flesken-Nikitin, Coulter Q. Ralston. AUTHORS AND AFFILIATIONS * Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA Andrea Flesken-Nikitin, 

Coulter Q. Ralston, Dah-Jiun Fu, Daryl J. Phuong, Blaine A. Harlan, Christopher S. Ashe, Amanda P. Armstrong, John C. Schimenti & Alexander Yu. Nikitin * Meinig School of Biomedical

Engineering, Cornell University, Ithaca, NY, USA Coulter Q. Ralston, David W. McKellar & Benjamin D. Cosgrove * Department of Oncology and Children’s Research Center, University

Children’s Hospital Zürich, Zürich, Switzerland Andrea J. De Micheli * Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Daryl J. Phuong, Sangeeta Ghuwalewala

 & John C. Schimenti * Memorial Sloan Kettering Cancer Center, New York, NY, USA Lora H. Ellenson Authors * Andrea Flesken-Nikitin View author publications You can also search for this

author inPubMed Google Scholar * Coulter Q. Ralston View author publications You can also search for this author inPubMed Google Scholar * Dah-Jiun Fu View author publications You can also

search for this author inPubMed Google Scholar * Andrea J. De Micheli View author publications You can also search for this author inPubMed Google Scholar * Daryl J. Phuong View author

publications You can also search for this author inPubMed Google Scholar * Blaine A. Harlan View author publications You can also search for this author inPubMed Google Scholar * Christopher

S. Ashe View author publications You can also search for this author inPubMed Google Scholar * Amanda P. Armstrong View author publications You can also search for this author inPubMed 

Google Scholar * David W. McKellar View author publications You can also search for this author inPubMed Google Scholar * Sangeeta Ghuwalewala View author publications You can also search

for this author inPubMed Google Scholar * Lora H. Ellenson View author publications You can also search for this author inPubMed Google Scholar * John C. Schimenti View author publications

You can also search for this author inPubMed Google Scholar * Benjamin D. Cosgrove View author publications You can also search for this author inPubMed Google Scholar * Alexander Yu.

Nikitin View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.F.N. and A.Y.N. designed experiments; C.Q.R., D.J.F., D.J.P., B.A.H., C.S.A.,

A.P.A., and S.G. performed experiments; C.Q.R., A.J.D.M., D.W.M., and B.D.C. carried our bioinformatics analyses; D.J.F., L.H.E., and A.Y.N. performed pathological evaluations; J.C.S., and

B.D.C. provided resources; A.F.N., C.Q.R. and A.Y.N. wrote the paper. CORRESPONDING AUTHOR Correspondence to Alexander Yu. Nikitin. ETHICS DECLARATIONS COMPETING INTERESTS The authors

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