Neoadjuvant chemotherapy induces phenotypic mast cell changes in high grade serous ovarian cancer

Abstract

Background: High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy in which patients have still yet to respond meaningfully to clinically available immunotherapies. Hence, novel immune targets are urgently needed. Our past work has identified that mast cells are significantly upregulated at the mRNA level in HGSOC patient tumors following neoadjuvant chemotherapy (NACT) exposure. Therefore, in this current investigation we sought to characterize intratumoral mast cell phenotypic changes as a result of NACT exposure and determine how these adaptations are associated with patient clinical outcomes.

Methods: Hematologic immunohistochemistry was employed to determine mast cell levels in 36 matched pre- and post-NACT HGSOC patient tumors. Fluorescent Immunohistochemistry was utilized to identify Tryptase+(carboxypeptidase A3 (CPA3) + mast cells as well as histamine levels in 29 and 20, respectively, matched pre- and post-NACT HGSOC patient tumors. Finally, human immortalized mast cells, LUVA were stimulated with carboplatin and paclitaxel and genomic changes were analyzed by quantitative PCR.

Results: Hematologic labeled intratumoral mast cells were significantly upregulated in the intraepithelial and stromal regions of the tumor, post-NACT. Lower levels of pre-NACT mast cells were significantly associated with an improved progression-free survival (PFS). Histamine, a marker of mast cell degranulation was similarly upregulated in post-NACT exposed tumors. Through the characterization of mast cell specific proteases Tryptase and CPA3, it was found that Tryptase+/ CPA3 + mast cells were significantly upregulated both in the intraepithelial and stromal compartments of the tumor, while Tryptase + cells were significantly upregulated in the stromal regions of the tumor. Lower post-NACT treated levels with Tryptase+/ CPA3 + cells were significantly associated with improved overall survival (OS) and PFS while higher Tryptase + mast cells were associated with improved OS. Finally, following chemotherapy exposure mast cell activating factors AREG and CCL2 were significantly upregulated while TGFB1, an inhibitor of mast cell activation was downregulated in LUVA cells.

Conclusions: Enhanced mast cell numbers, as well as activation and degranulation are a consequence of NACT exposure. Post-NACT mast cells displayed differing associations with survival outcomes that was dependent upon granule classification. Ultimately, mast cells represent a clinically relevant putative HGSOC immune target.

Keywords: High grade serous ovarian cancer; Mast cells; Neoadjuvant chemotherapy.

Fig. 1

Intratumoral stromal and intraepithelial mast cells are upregulated following chemotherapy exposure. (A) Representative images of hematologic mast cell staining in 36 matched pre- and post-NACT HGSOC patient tumors. Mast cells were depicted by a bright blue stain, while eosinophils were stained bright red and nuclei of cells a dull blue. Average number of positive (B) total and (C) intraepithelial and stromal mast cells pre- and post-NACT. Statistically significant differences determined by Student T-test. NACT, neoadjuvant chemotherapy, *p < 0.05, as indicated

Fig. 2

Patient survival outcomes associated with pre-treatment hematologic mast cell levels. Kaplan-Meier curve analysis of pre-treatment (A) total (B) intraepithelial mast cells association with progression free survival stratified by lower and upper quartile. Kaplan-Meier curve analysis of pre-treatment (C) total and (D) intraepithelial mast cells association with overall survival stratified by lower and upper quartile. HR and associated log-rank p-values were reported. HR, hazard ratio

Fig. 3

Intratumoral histamine is upregulated following chemotherapy exposure (A) Representative immunofluorescence images of histamine expression in 20 matched pre- and post-NACT tumors and corresponding secondary only, negative control taken at a 10x objective. (B) Mean intensity (pixels) and (C) Integrated optical density of histamine in pre- versus post-NACT HGSOC tumors. Statistically significant differences determined by Student T-test. NACT, neoadjuvant chemotherapy, HGSOC, high grade serous ovarian cancer, *p < 0.05, as indicated

Fig. 4

Tryptase +/ CPA3 + mast cells are upregulated following chemotherapy exposure (A) Representative immunofluorescence images of Tryptase, CPA3 expression in 29 matched pre- and post-NACT tumors and corresponding secondary only, negative control taken at a 40x objective. (B) Average number of Tryptase + /CPA3 + cells per field in matched pre-and post-NACT exposed HGSOC tumors. Statistically significant differences determined by Student T-test. NACT, neoadjuvant chemotherapy, HGSOC, high grade serous ovarian cancer, ***p < 0.0005, as indicated

Fig. 5

Tryptase+/CPA3 + and Tryptase + cell subset expression in pre-and post-NACT HGSOC tumors. (A) intraepithelial and (B) stromal Tryptase+/CPA3 + mast cells in 29 matched pre- and post- NACT treated tumors. (C) total, (D) stomal, and (E) intraepithelial Tryptase + mast cells in 29 matched pre- and post- NACT treated tumors. Statistically significant differences determined by Student T-test. NACT, neoadjuvant chemotherapy, HGSOC, high grade serous ovarian cancer, * p < 0.05, ***p < 0.0005, ****p < 0.00005, as indicated, ns, non-significant

Fig. 6

Tryptase+/CPA3 + mast cells association with overall survival. Kaplan-Meier survival curve analysis of post-NACT (A) total, (B) intraepithelial, and (C) stromal Tryptase/CPA3 + mast cells association with overall survival stratified by median levels. HR and associated log-rank p-values were reported. NACT, neoadjuvant chemotherapy; HR, hazard ratio

Fig. 7

Tryptase+/CPA3 + mast cells association with progression-free survival. Post-NACT (A) total, (B) intraepithelial, and (C) stromal Tryptase+/CPA3 + levels stratified by progression-free survival 12-month cut-off. Statistically significant differences determined by Student T-test. NACT, neoadjuvant chemotherapy, *p < 0.05, as indicated, ns, non-significant

Fig. 8

Kaplan-Meier survival curve analysis of total post-NACT Tryptase + mast cells association with overall survival stratified by upper and lower quartile expression. HR and associated log-rank p-values were reported. NACT, neoadjuvant chemotherapy; HR, hazard ratio

Fig. 9

qPCR analysis of LUVA mast cells exposed to standard of care HGSOC chemotherapy. (A) AREG, (B) CCL2, (C)TGFβ1, (D) VEGFA, (E) TNF, (F) IL-2, (G) CCL7, (H) IL-10, and (I) TGFβ2 mRNA levels in LUVA cells stimulated with 100 μm of carboplatin and 10 nm of paclitaxel for 48h and analyzed via qPCR. Error bars represent standard deviation of ≥ 3 biological replicates. Statistically significant differences determined by Student T-test. NACT, neoadjuvant chemotherapy, HGSOC, high grade serous ovarian cancer, *p < 0.05, ** p < 0.005, as indicated, ns, non-significant