Clinical outcomes following identification of an incidental p53 signature in the fallopian tube.

Fallopian tube pathology in patients with BRCA1 and BRCA2 mutations suggests a possible pathway to high grade serous ovarian carcinoma originates with a p53 signature, which is thought to represent a potential precursor to serous tubal intraepithelial carcinoma (STIC). The clinical implications of an isolated p53 signature in the average-risk population has not been well-established. This study aims to describe clinical outcomes in patients with incidentally noted p53 signature lesions. All patients diagnosed with a p53 signature lesion on final pathology from 2014 to 2022 were identified at a large academic institution. P53 signature is defined by our lab as morphologically normal to mildly atypical tubal epithelium with focal p53 over-expression on immunohistochemistry. Incidental p53 signature was defined as identification of a fallopian tube lesion excised for benign or unrelated indications in patients without a known hereditary disposition. Demographic, clinicopathologic, and genetic data were collected. A total of 127 patients with p53 signatures were identified. Thirty-six patients were excluded for established ovarian cancer or high-risk history leaving 91 total patients. Five patients (5.5%) developed a malignancy, none of which were ovarian or primary peritoneal, at the end of the eight and a half year follow up period. Twenty-four (26.4%) patients had salpingectomy without any form of oophorectomy at the time of initial surgery, while 67 (73.6%) patients had at least a unilateral oophorectomy at the time of their salpingectomy. Seven patients (7.7%) had additional surgery after p53 signature diagnosis; however, the final pathology yielded no evidence of malignancy in all these patients. After subsequent surgeries, 19 (20.9%) patients maintained their ovaries. The diagnosis of an incidental p53 signature was not associated with any primary peritoneal or ovarian cancer diagnoses during our follow up, and the majority of patients were managed conservatively by their providers with no further intervention after diagnosis.

Development of substituted benzimidazoles as inhibitors of human aldehyde dehydrogenase 1A isoenzymes.

Aldehyde dehydrogenase 1A (ALDH1A) isoforms may be a useful target for overcoming chemotherapy resistance in high-grade serous ovarian cancer (HGSOC) and other solid tumor cancers. However, as different cancers express different ALDH1A isoforms, isoform selective inhibitors may have a limited therapeutic scope. Furthermore, resistance to an ALDH1A isoform selective inhibitor could arise via induction of expression of other ALDH1A isoforms. As such, we have focused on the development of pan-ALDH1A inhibitors, rather than on ALDH1A isoform selective compounds. Herein, we report the development of a new group of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in HGSOC. Optimization of the CM10 scaffold, aided by ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular efficacy as demonstrated by reduction in ALDEFLUOR signal in HGSOC cells, and substantial improvements in liver microsomal stability. Based on this work we identified two compounds 17 and 25 suitable for future in vivo proof of concept experiments.

EGFL6 promotes endometrial cancer cell migration and proliferation.

OBJECTIVE: EGFL6, a growth factor produced by adipocytes, is upregulated in and implicated in the tumorigenesis of multiple tumor types. Given the strong link between obesity and endometrial cancer, we sought to determine the impact of EGFL6 on endometrial cancer. METHODS: EGFL6 expression in endometrial cancer and correlation with patient outcomes was evaluated in the human protein atlas and TCGA. EGFL6 treatment, expression upregulation, and shRNA knockdown were used to evaluate the impact of EGFL6 on the proliferation and migration of 3 endometrial cancer cell lines in vitro. Similarly, the impact of EGFL6 expression and knockdown on tumor growth was evaluated. Western blotting was used to evaluate the impact of EGFL6 on MAPK phosphorylation. RESULTS: EGFL6 is upregulated in endometrial cancer, primarily in cony-number high tumors. High tumor endometrial cancer expression of EGFL6 predicts poor patient prognosis. We find that EGFL6 acts to activate the MAPK pathway increasing cellular proliferation and migration. In xenograft models, EGFL6 overexpression increases endometrial cancer tumor growth while EGFL6 knockdown decreases endometrial cancer tumor growth. CONCLUSIONS: EGFL6 is a marker of poor prognosis endometrial cancers, driving cancer cell proliferation and growth. As such EGFL6 represents a potential therapeutic target in endometrial cancer.

Single-cell analysis reveals the stromal dynamics and tumor-specific characteristics in the microenvironment of ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC) is a heterogeneous disease, and a highstromal/desmoplastic tumor microenvironment (TME) is associated with a poor outcome. Stromal cell subtypes, including fibroblasts, myofibroblasts, and cancer-associated mesenchymal stem cells, establish a complex network of paracrine signaling pathways with tumor-infiltrating immune cells that drive effector cell tumor immune exclusion and inhibit the antitumor immune response. In this work, we integrate single-cell transcriptomics of the HGSOC TME from public and in-house datasets (n = 20) and stratify tumors based upon high vs. low stromal cell content. Although our cohort size is small, our analyses suggest a distinct transcriptomic landscape for immune and non-immune cells in high-stromal vs. low-stromal tumors. High-stromal tumors have a lower fraction of certain T cells, natural killer (NK) cells, and macrophages, and increased expression of CXCL12 in epithelial cancer cells and cancer-associated mesenchymal stem cells (CA-MSCs). Analysis of cell-cell communication indicate that epithelial cancer cells and CA-MSCs secrete CXCL12 that interacte with the CXCR4 receptor, which is overexpressed on NK and CD8+ T cells. Dual IHC staining show that tumor infiltrating CD8 T cells localize in proximity of CXCL12+ tumor area. Moreover, CXCL12 and/or CXCR4 antibodies confirm the immunosuppressive role of CXCL12-CXCR4 in high-stromal tumors.

A putative role for ALDH inhibitors and chemoprevention of BRCA-mutation-driven tumors.

Aldehyde dehydrogenase (ALDH) enzymatic activity is a marker of cancer-initiating cells (CIC) in many tumor types. Our group and others have found that ALDH1A family inhibitors (ALDHi) can preferentially induce death of ovarian CIC in established ovarian cancer. We sought to determine if ALDHi, by targeting CIC at the time of tumor initiation, could function as a chemopreventive for ovarian cancer. As BRCA1/2 mutation carriers represent a population who could benefit from an ovarian cancer chemopreventive, we focused on BRCA mutation-associated tumor cell lines and murine tumor models. We found that, compared to BRCA wild-type cells, BRCA mutant ovarian cancer cells are more sensitive to the ALDHi673A. Similarly, while 673A treatment of wild-type fallopian tube epithelial (FTE) cells is non-toxic, 673A induces death in FTE cells with BRCA1 knockdown. Using a murine fallopian tube organoid model of ovarian carcinogenesis, we show that 673A reduced organoid complexity and significantly reduce colony formation of BRCA-mutant cells. Organoids that persisted after 673A treatment were predominantly BRCA1wt, but NF1 mutant, suggesting a resistance mechanism. Finally, using the BPRN (Brca1, Trp53, Rb1, Nf1 inactivated) mouse model of tubo-ovarian cancer, we evaluated the impact of intermittent 673A therapy on carcinogenesis. 673A treatment resulted in a significant reduction in serous tubal intraepithelial carcinoma (STIC) lesions and carcinomas. Collectively, the findings suggest that ALDHi, such as 673A, could serve as chemopreventive agents for BRCA1/2 mutation carriers.

Evaluating the trophic transfer of PCBs from fish to humans: Insights from a synergism of environmental monitoring and physiologically-based pharmacokinetic modeling.

Accumulation of polychlorinated biphenyls (PCBs) within fish tissues has prompted many states to issue consumption advisories. In Pennsylvania such advisories suggest one meal per month for most game species harvested from Lake Erie; however, these advisories do not account for the emergent properties of regional PCB mixtures, and the downstream accumulation of PCB congeners into human tissues is poorly documented. This study aimed to demonstrate the utility of pairing environmental monitoring with pharmacokinetic modeling for the purpose of estimating dietary PCB exposure in humans. We qualified and quantified the PCB congeners present in the filets of five Lake Erie fish species and used these data to estimate exposure under consumption scenarios that matched or exceeded the advisories. Physiologically-based pharmacokinetic (PBPK) modeling was then employed to predict PCB accumulation within seven tissue compartments of a hypothetical man and woman over 10 years. Twenty-one congeners were detected between the five fish species at concentrations ranging from 56.0 to 411.7 ng/g. Predicted accumulation in human tissues varied based on tissue type, the species consumed, biological sex, and fish-consumption rate. Notably, steady-state concentrations were higher in fatty tissue compartments ("Fat" and "Liver") and across all tissues in women compared to men. This study serves as a preliminary blueprint for generating predictions of site-specific and tissue-specific exposure through the integration of environmental monitoring and pharmacokinetic modeling. Although the details may vary across applications, this simple approach could complement traditional exposure assessments for vulnerable communities in the Great Lakes region that continue to suffer from legacy contamination.

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies.

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of Significance: We developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

Tumor-Associated Macrophages Expand Chemoresistant, Ovarian Cancer Stem-Like Cells.

The persistence of ovarian cancer stem-like cells (OvCSCs) after chemotherapy resistance has been implicated in relapse. However, the ability of these relatively quiescent cells to produce the robust tumor regrowth necessary for relapse remains an enigma. Since normal stem cells exist in a niche, and tumor-associated macrophages (TAMs) are the highest abundance immune cell within ovarian tumors, we hypothesized that TAMs may influence OvCSC proliferation. To test this, we optimized OvCSC enrichment by sphere culture and in vitro polarization of monocytes to a TAM-like M2 phenotype. Using cocultures that permitted the exchange of only soluble factors, we found that M2 macrophages increased the proliferation of sphere cells. Longer-term exposure (5-7 days) to soluble TAM factors led to retention of some stem cell features by OvCSCs but loss of others, suggesting that TAMs may support an intermediate stemness phenotype in OvCSCs. Although TAM coculture decreased the percentage of OvCSCs surviving chemotherapy, it increased the overall number. We therefore sought to determine the influence of this interaction on chemotherapy efficacy in vivo and found that inhibiting macrophages improved chemotherapy response. Comparing the gene expression changes in OvCSCs cocultured with TAMs to publicly available patient data identified 34 genes upregulated in OvCSCs by exposure to soluble TAM factors whose expression correlates with outcome. Overall, these data suggest that TAMs may influence OvCSC proliferation and impact therapeutic response.

Single-cell analysis reveals the stromal dynamics and tumor-specific characteristics in the microenvironment of ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC) is a heterogeneous disease, and a high stromal/desmoplastic tumor microenvironment (TME) is associated with a poor outcome. Stromal cell subtypes, including fibroblasts, myofibroblasts, and cancer-associated mesenchymal stem cells, establish a complex network of paracrine signaling pathways with tumor-infiltrating immune cells that drive effector cell tumor immune exclusion and inhibit the antitumor immune response. Single-cell transcriptomics of the HGSOC TME from public and in-house datasets revealed a distinct transcriptomic landscape for immune and non-immune cells in high-stromal vs. low-stromal tumors. High-stromal tumors had a lower fraction of certain T cells, natural killer (NK) cells, and macrophages and increased expression of CXCL12 in epithelial cancer cells and cancer-associated mesenchymal stem cells (CA-MSCs). Analysis of cell-cell communication indicated that epithelial cancer cells and CA-MSCs secreted CXCL12 that interacted with the CXCR4 receptor, which was overexpressed on NK and CD8 + T cells. CXCL12 and/or CXCR4 antibodies confirmed the immunosuppressive role of CXCL12-CXCR4 in high-stromal tumors.

Generation and characterization of humanized affinity-matured EGFL6 antibodies for ovarian cancer therapy.

OBJECTIVES: Epidermal growth factor EGF-like domain multiple-6 (EGFL6) is highly expressed in high grade serous ovarian cancer and promotes both endothelial cell proliferation/angiogenesis and cancer cell proliferation/metastasis. As such it has been implicated as a therapeutic target. As a secreted factor, EGFL6 is a candidate for antibody therapy. The objectives of this study were to create and validate humanized affinity-matured EGFL6 neutralizing antibodies for clinical development. METHODS: A selected murine EGFL6 antibody was humanized using CDR grafting to create 26 variant humanized antibodies. These were screened and the lead candidate was affinity matured. Seven humanized affinity-matured EGFL6 antibodies were screened for their ability to block EGFL6 activity on cancer cells in vitro, two of which were selected and tested their therapeutic activity in vivo. RESULTS: Humanized affinity matured antibodies demonstrated high affinity for EGFL6 (150 pM to 2.67 nM). We found that several humanized affinity-matured EGFL6 antibodies specifically bound to recombinant, and native human EGFL6. Two lead antibodies were able to inhibit EGFL6-mediated (i) cancer cell migration, (ii) proliferation, and (iii) increase in ERK phosphorylation in cancer cells in vitro. Both lead antibodies restricted growth of an EGFL6 expressing ovarian cancer patient derived xenograft. Analysis of treated human tumor xenografts indicated that anti-EGFL6 therapy suppressed angiogenesis, inhibited tumor cell proliferation, and promoted tumor cell apoptosis. CONCLUSIONS: Our studies confirm the ability of these humanized affinity-matured antibodies to neutralize EGFL6 and acting as a therapeutic to restrict cancer growth. This work supports the development of these antibody for first-in-human clinical trials.

Quiescent Ovarian Cancer Cells Secrete Follistatin to Induce Chemotherapy Resistance in Surrounding Cells in Response to Chemotherapy.

PURPOSE: We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase ovarian cancer chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4-driven ovarian cancer chemoresistance. EXPERIMENTAL DESIGN: We used RNA sequencing to identify NFATC4-mediated differential gene expression. CRISPR-Cas9 and FST (follistatin)-neutralizing antibodies were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy. RESULTS: We found that NFATC4 upregulates FST mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2-dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR knockout (KO) of FST in ovarian cancer cells or antibody-mediated neutralization of FST sensitizes ovarian cancer cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy-resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of patients with ovarian cancer significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression-free, post-progression-free, and overall survival. CONCLUSIONS: FST is a novel therapeutic target to improve ovarian cancer response to chemotherapy and potentially reduce recurrence rates.

Clinical factors associated with failed sentinel lymph node mapping in endometrial cancer.

Objective: Sentinel lymph node (SLN) mapping is a highly accurate surgical technique for detecting metastases in endometrial cancer. The objective of this study was to identify clinical factors associated with failed mapping. Methods: All patients with endometrial cancer undergoing minimally-invasive staging and planned SLN biopsy from 1/1/2017 to 12/31/2020 at a single institution were identified retrospectively. Demographic, clinicopathologic and treatment data were obtained. Data were compared using descriptive statistics. Univariate and multivariable logistic regression were performed to identify predictors of failed mapping. Results: 819 patients were identified with a mean age of 64.6 years (range 26-93) and mean BMI of 35.6 kg/m2 (range 18-68). Most (88.5 %, 725/819) had early-stage disease and endometrioid histology (82.3 %, 674/819). A majority (74.2 %, 608/819) had successful bilateral mapping, and 54 (6.6 %) had unsuccessful bilateral mapping. Increasing BMI was significantly associated with unsuccessful bilateral mapping: patients with BMI > 30 were more likely to have unsuccessful SLN mapping (p = 0.033). Among patients with known lymph node status (799/819), patients with macrometastases and micrometastases were more likely to have failed bilateral mapping compared to those with negative SLNs or isolated tumor cells (p = 0.013). On multivariable analysis, higher BMI and histology were associated with failed bilateral mapping (OR = 1.023, 95 % CI (1.005, 1.041) and OR = 1.678, 95 % CI (1.177, 2.394), respectively). Conclusion: SLN mapping has a high success in patients undergoing minimally-invasive surgical staging for endometrial cancer. Increasing BMI, high risk histology, and lymph node metastases are risk factors for failed mapping.

Phase I trial of ribociclib with platinum chemotherapy in ovarian cancer.

BACKGROUNDNew therapeutic combinations to improve outcomes of patients with ovarian cancer are clearly needed. Preclinical studies with ribociclib (LEE-011), a CDK4/6 cell cycle checkpoint inhibitor, demonstrate a synergistic effect with platinum chemotherapy and efficacy as a maintenance therapy after chemotherapy. We tested the safety and initial efficacy of ribociclib in combination with platinum-based chemotherapy in recurrent ovarian cancer.METHODSThis phase I trial combined weekly carboplatin and paclitaxel chemotherapy with ribociclib, followed by ribociclib maintenance in patients with recurrent platinum-sensitive ovarian cancer. Primary objectives were safety and maximum tolerated dose (MTD) of ribociclib when given with platinum and taxane chemotherapy. Secondary endpoints were response rate (RR) and progression-free survival (PFS).RESULTSThirty-five patients were enrolled. Patients had a mean of 2.5 prior lines of chemotherapy, and 51% received prior maintenance therapy with poly(ADP-ribose) polymerase inhibitors and/or bevacizumab. The MTD was 400 mg. The most common adverse events included anemia (82.9%), neutropenia (82.9%), fatigue (82.9%), and nausea (77.1%). The overall RR was 79.3%, with a stable disease rate of 18%, resulting in a clinical benefit rate of 96.6%. Median PFS was 11.4 months. RR and PFS did not differ based on the number of lines of prior chemotherapy or prior maintenance therapy.CONCLUSIONThis work demonstrates that the combination of ribociclib with chemotherapy in ovarian cancer is feasible and safe. With a clinical benefit rate of 97%, this work provides encouraging evidence of clinical efficacy in patients with recurrent platinum-sensitive disease.TRIAL REGISTRATIONClinicalTrials.gov NCT03056833.FUNDINGThis investigator-initiated trial was supported by Novartis, which provided drugs and funds for trial execution.

Targeting Therapeutic Resistance and Multinucleate Giant Cells in CCNE1-Amplified HR-Proficient Ovarian Cancer.

Approximately 20% of high-grade serous ovarian cancers (HGSOC) have CCNE1 amplification. CCNE1-amplified tumors are homologous recombination (HR) proficient and resistant to standard therapies. Therapy resistance is associated with increased numbers of polyploid giant cancer cells (PGCC). We sought to identify new therapeutic approaches for patients with CCNE1-amplified tumors. Using TCGA data, we find that the mTOR, HR, and DNA checkpoint pathways are enriched in CCNE1-amplified ovarian cancers. Furthermore, Interactome Mapping Analysis linked the mTOR activity with upregulation of HR and DNA checkpoint pathways. Indeed, we find that mTOR inhibitors (mTORi) downregulate HR/checkpoint genes in CCNE1-amplified tumors. As CCNE1-amplified tumors are dependent on the HR pathway for viability, mTORi proved selectively effective in CCNE1-amplified tumors. Similarly, via downregulation of HR genes, mTORi increased CCNE1-amplifed HGSOC response to PARPi. In contrast, overexpression of HR/checkpoint proteins (RAD51 or ATR), induced resistance to mTORi. In vivo, mTORi alone potently reduced CCNE1-amplified tumor growth and the combination of mTORi and PARPi increased response and tumor eradication. Tumors treated with mTORi demonstrated a significant reduction in ALDH+ PGCCs. Finally, as a proof of principle, we identified three patients with CCNE1 amplified tumors who were treated with an mTORi. All three obtained clinical benefits from the therapy. Our studies and clinical experience indicate mTORi are a potential therapeutic approach for patients with CCNE1-amplified tumors.

Shifting the Soil: Metformin Treatment Decreases the Protumorigenic Tumor Microenvironment in Epithelial Ovarian Cancer.

Controversy persists regarding metformin's role in cancer therapy. Our recent work suggested metformin acts by impacting the tumor microenvironment (TME), normalizing the epigenetic profile of cancer-associated mesenchymal stem cells (CA-MSC). As CA-MSC can negatively impact tumor immune infiltrates, we evaluated metformin's impact on the human TME, focusing on the interplay of stroma and immune infiltrates. Tumor samples from (i) 38 patients treated with metformin and chemotherapy and (ii) 44 non-metformin matched controls were included in a tissue microarray (TMA). The TMA was used to compare the presence of CA-MSC, desmoplasia and immune infiltrates in the TME. In vitro and in vivo models examined metformin's role in alteration of the CA-MSC phenotype. The average percentage of CA-MSC was significantly lower in metformin-treated than in chemotherapy alone-treated tumors (p = 0.006). There were fewer regulatory T-cells in metformin-treated tumors (p = 0.043). Consistent with CA-MSC's role in excluding T-cells from tumor islets, the T-cells were primarily present within the tumor stroma. Evaluation of metformin's impact in vitro suggested that metformin cannot reverse a CA-MSC phenotype; however, the in vivo model where metformin was introduced prior to the establishment of the CA-MSC phenotype supported that metformin can partially prevent the reprogramming of normal MSC into CA-MSC. Metformin treatment led to a decrease in both the presence of protumorigenic CA-MSC and in immune exclusion of T cells, leading to a more immune-permissive environment. This suggests clinical utility in prevention and in treatment for early-stage disease and putatively in immune therapy.

Intestinal Radiation Protection and Mitigation by Second-Generation Probiotic Lactobacillus-reuteri Engineered to Deliver Interleukin-22.

(1) Background: The systemic administration of therapeutic agents to the intestine including cytokines, such as Interleukin-22 (IL-22), is compromised by damage to the microvasculature 24 hrs after total body irradiation (TBI). At that time, there is significant death of intestinal microvascular endothelial cells and destruction of the lamina propria, which limits drug delivery through the circulation, thus reducing the capacity of therapeutics to stabilize the numbers of Lgr5+ intestinal crypt stem cells and their progeny, and improve survival. By its direct action on intestinal stem cells and their villus regeneration capacity, IL-22 is both an ionizing irradiation protector and mitigator. (2) Methods: To improve delivery of IL-22 to the irradiated intestine, we gavaged Lactobacillus-reuteri as a platform for the second-generation probiotic Lactobacillus-reuteri-Interleukin-22 (LR-IL-22). (3) Results: There was effective radiation mitigation by gavage of LR-IL-22 at 24 h after intestinal irradiation. Multiple biomarkers of radiation damage to the intestine, immune system and bone marrow were improved by LR-IL-22 compared to the gavage of control LR or intraperitoneal injection of IL-22 protein. (4) Conclusions: Oral administration of LR-IL-22 is an effective protector and mitigator of intestinal irradiation damage.

Carcinoma-Associated Mesenchymal Stem/Stromal Cells: Architects of the Pro-tumorigenic Tumor Microenvironment.

The interaction between tumor cells and non-malignant hosts cells within the tumor microenvironment (TME) is critical to the pathophysiology of cancer. These non-malignant host cells, consisting of a variety of stromal, immune, and endothelial cells, engage in a complex bidirectional crosstalk with the malignant tumor cells. Mesenchymal stem/stromal cells (MSCs) are one of these host cells, and they play a critical role in directing the formation and function of the entire TME. These MSCs are epigenetically reprogrammed by cancer cells to assume a strongly pro-tumorigenic phenotype and are referred to as carcinoma-associated mesenchymal stem/stromal cells (CA-MSCs). Studies over the last decade demonstrate that CA-MSCs not only directly interact with cancer cells to promote tumor growth and metastasis but also orchestrate the formation of the TME. Carcinoma-associated mesenchymal stem/stromal cells can differentiate into virtually all stromal sub-lineages present in the TME, including pro-tumorigenic cancer-associated fibroblasts (CAF), myofibroblasts, and adipocytes. carcinoma-associated mesenchymal stem/stromal cells and the CAFs they produce, secrete much of the extracellular matrix in the TME. Furthermore, CA-MSC secreted factors promote angiogenesis, and recruit immunosuppressive myeloid cells effectively driving tumor immune exclusion. Thus CA-MSCs impact nearly every aspect of the TME. Despite their influence on cancer biology, as CA-MSCs represent a heterogenous population without a single definitive marker, significant confusion remains regarding the origin and proper identification CA-MSCs. This review will focus on the impact of CA-MSCs on cancer progression and metastasis and the ongoing work on CA-MSC identification, nomenclature and mechanism of action.

Metformin and survival: Is there benefit in a cohort limited to diabetic women with endometrial, breast, or ovarian cancer?

OBJECTIVE: Evaluate the association between metformin and survival in women with Type 2 diabetes (T2DM) and breast, endometrial and ovarian cancer- 3 hormonally mediated cancers. METHODS: We evaluated outcomes in a cohort of 6225 women with T2DM with a new diagnosis of ovarian, breast or endometrial cancer from 2010 to 2019. We classified glycemic medications at time of first cancer diagnosis into 3 tiers in accordance with ADA guidelines. Approaches compared: (i) metformin (tier 1) vs. no glycemic medication, (ii) metformin vs tier 2 medications (sulfonylureas, thiazolidinediones, SGLT2-inhibitors, DPP4-inhibitors, alpha glucosidase-inhibitors, GLP-1 agonists), (iii) metformin vs tier 3 medications (insulins, amylinomimetics), and (iv) tier 2 vs tier 3 medications. Analyses included Cox proportional-hazards models, Kaplan-Meier curves, and conditional logistic regression in a risk set-sampled nested case-control matched on T2DM duration- all modeling survival. Models were adjusted for demographics, cancer type, A1C, T2DM duration, and number of office visits and hospitalizations. RESULTS: Metformin was the most used medication (n = 3232) and consistently demonstrated survival benefit compared with tier 2 and 3 medications, across all methods. Tier 3-users demonstrated highest risk of death when compared to metformin rather than tier 2 [adjHR = 1.83 (95% CI: 1.58, 2.13) vs. adjHR = 1.32 (95% CI: 1.11, 1.57)], despite similar baseline profiles between tier 1 and 2 users. CONCLUSIONS: Metformin users experienced increased survival even after accounting for surrogates of diabetes progression. Benefit extended beyond that seen in tier 2-users. Our findings, consistent with prior studies, indicate metformin use improves survival in women with T2DM and hormonally mediated women's cancers.

Cancer-associated MSC drive tumor immune exclusion and resistance to immunotherapy, which can be overcome by Hedgehog inhibition.

We investigated the impact of cancer-associated mesenchymal stem cells (CA-MSCs) on ovarian tumor immunity. In patient samples, CA-MSC presence inversely correlates with the presence of intratumoral CD8+ T cells. Using an immune "hot" mouse ovarian cancer model, we found that CA-MSCs drive CD8+ T cell tumor immune exclusion and reduce response to anti­PD-L1 immune checkpoint inhibitor (ICI) via secretion of numerous chemokines (Ccl2, Cx3cl1, and Tgf-ß1), which recruit immune-suppressive CD14+Ly6C+Cx3cr1+ monocytic cells and polarize macrophages to an immune suppressive Ccr2hiF4/80+Cx3cr1+CD206+ phenotype. Both monocytes and macrophages express high levels of transforming growth factor ß­induced (Tgfbi) protein, which suppresses NK cell activity. Hedgehog inhibitor (HHi) therapy reversed CA-MSC effects, reducing myeloid cell presence and expression of Tgfbi, increasing intratumoral NK cell numbers, and restoring response to ICI therapy. Thus, CA-MSCs regulate antitumor immunity, and CA-MSC hedgehog signaling is an important target for cancer immunotherapy.

The MEK1/2 Pathway as a Therapeutic Target in High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is the deadliest of gynecological cancers due to its high recurrence rate and acquired chemoresistance. RAS/MEK/ERK pathway activation is linked to cell proliferation and therapeutic resistance, but the role of MEK1/2-ERK1/2 pathway in HGSOC is poorly investigated. We evaluated MEK1/2 pathway activity in clinical HGSOC samples and ovarian cancer cell lines using immunohistochemistry, immunoblotting, and RT-qPCR. HGSOC cell lines were used to assess immediate and lasting effects of MEK1/2 inhibition with trametinib in vitro. Trametinib effect on tumor growth in vivo was investigated using mouse xenografts. MEK1/2 pathway is hyperactivated in HGSOC and is further stimulated by cisplatin treatment. Trametinib treatment causes cell cycle arrest in G1/0-phase and reduces tumor growth rate in vivo but does not induce cell death or reduce fraction of CD133+ stem-like cells, while increasing expression of stemness-associated genes instead. Transient trametinib treatment causes long-term increase in a subpopulation of cells with high aldehyde dehydrogenase (ALDH)1 activity that can survive and grow in non-adherent conditions. We conclude that MEK1/2 inhibition may be a promising approach to suppress ovarian cancer growth as a maintenance therapy. Promotion of stem-like properties upon MEK1/2 inhibition suggests a possible mechanism of resistance, so a combination with CSC-targeting drugs should be considered.