Multiparameter single-cell proteomic technologies give new insights into the biology of ovarian tumors

Abstract

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. Its diagnosis at advanced stage compounded with its excessive genomic and cellular heterogeneity make curative treatment challenging. Two critical therapeutic challenges to overcome are carboplatin resistance and lack of response to immunotherapy. Carboplatin resistance results from diverse cell autonomous mechanisms which operate in different combinations within and across tumors. The lack of response to immunotherapy is highly likely to be related to an immunosuppressive HGSOC tumor microenvironment which overrides any clinical benefit. Results from a number of studies, mainly using transcriptomics, indicate that the immune tumor microenvironment (iTME) plays a role in carboplatin response. However, in patients receiving treatment, the exact mechanistic details are unclear. During the past decade, multiplex single-cell proteomic technologies have come to the forefront of biomedical research. Mass cytometry or cytometry by time-of-flight, measures up to 60 parameters in single cells that are in suspension. Multiplex cellular imaging technologies allow simultaneous measurement of up to 60 proteins in single cells with spatial resolution and interrogation of cell-cell interactions. This review suggests that functional interplay between cell autonomous responses to carboplatin and the HGSOC immune tumor microenvironment could be clarified through the application of multiplex single-cell proteomic technologies. We conclude that for better clinical care, multiplex single-cell proteomic technologies could be an integral component of multimodal biomarker development that also includes genomics and radiomics. Collection of matched samples from patients before and on treatment will be critical to the success of these efforts.

Keywords: Carboplatin resistance; Immune tumor microenvironment; Mass cytometry/CyTOF; Multimodal biomarkers; Multiplex cellular imaging; Ovarian cancer; Single cell.

Fig. 1

Clinical management of HGSOC: diagnosis, treatment, and response. Diagnosis is usually at advanced stage. A total of 95% cases harbor mutations in TP53. A subgroup of tumors harbor mutations in BRCA1 and BRCA2 (~ 20%) or defects in other genes involved in homologous recombination repair (~ 30%). Patients with newly diagnosed HGSOC are treated either with upfront debulking surgery followed by chemotherapy (usually six cycles of carboplatin plus paclitaxel) or first with three cycles of neoadjuvant chemotherapy, followed by surgery and three additional cycles of chemotherapy. Molecular classification determines response to PARPi shown by shades of green (dark to lighter shades depict long to short PFS). HRD, homologous recombination deficient; HRP, homologous recombination proficient; CNA, copy number alteration; PARPi, poly (ADP ribose) polymerase inhibitor; PFS, progression-free survival; OS, overall survival

Fig. 2

CyTOF reveals heterogeneity of PARP1 and pH2AX protein levels in OVCAR3 cells. OVCAR3 cells were treated with carboplatin (8 µM) or DMSO (control) for 48 h. Cells were stained with CyTOF antibodies against DDR and cell cycle proteins. After normalization, the two CyTOF datasets were concatenated and analyzed by UMAP embedding, a dimensionality reduction algorithm using DDR and cell cycle protein levels. UMAP colored to depict A treatment (carboplatin or control), B cell cycle, C PARP1 expression, D pH2AX expression (a surrogate marker for HRD)

Fig. 3

Multiplex image showing micro-heterogeneity of an HGSOC tumor sample after NACT. This tumor exemplifies the existence of several iTMEs within a single tumor sample. Enlarged images reveal different iTMEs. (A-a) “Hot/inflamed” iTME: tumor cells (cytokeratin) infiltrated with T cells (CD3), macrophages (CD68), and B cells (CD19). (A-b) Mixed iTMEs: this part of the tumor shows areas that are “hot/inflamed”’ and “cold/excluded or desert”. (A-c) “Cold” iTME: abundance of macrophages with spatially distinct T cells with absence of tumor cells. (A-d) “Cold/excluded” iTME: tumor-rich region with non-infiltrating immune cells. (B) Schematic showing mechanisms reported to shape the HGSOC iTME. TAM, tumor-associated macrophage; IFNs, interferons; FBI, foldback inversions

Fig. 4

Multimodal biomarkers—personalized medicine for women with HGSOC. A multimodal biomarker comprised of data from radiomics, genomics, CyTOF, and multiplex imaging. Fluorescence-based multiplex imaging shown as a representative imaging platform. Circos plot of whole genome sequencing of DNA from HGSOC patient with CCNE1 amplification and HRP