Gierke, B. | Search bioRxiv

bioRxiv2023

ZeptoCTC - Sensitive Protein Analysis of True Single Cell Lysates using Reverse Phase Protein Arrays (RPPA)

Circulating Tumor Cells (CTCs) are commonly analyzed through genomic profiling, which does not capture posttranslational and functional alterations of encoded proteins. To address this limitation, we developed ZeptoCTC, a single-cell protein analysis workflow that combines established technologies for single-cell isolation and sensitive Reverse Phase Protein Array (RPPA) analysis to assess multiple protein expression and activation in individual CTCs. The workflow involves single cell labeling, isolation, lysis, and printing of the true single cell lysates onto a ZeptoChip using a modified micromanipulator CellCelectorTM. Subsequently, the printed lysates undergo fluorescence immunoassay RPPA protein detection using a ZeptoReader followed by signal quantification with Image J software. ZeptoCTC was successfully optimized, beginning with the measurement of EpCAM protein expression--a standard marker for CTC detection. As expected, mean fluorescence signals for EpCAM levels were significantly higher in single MCF-7 cells compared to MDA-MB-231 cells. Next, Capivasertib-treated MCF-7 cells exhibited an approximately 2-fold increase in the pAkt/Akt ratio compared to non-treated control cells. This finding was consistent with a co-performed western blot analysis of pooled MCF-7 cells. Application of ZeptoCTC to the analysis of single CTCs derived from a metastasized breast cancer (MBC) patient indicated a significantly higher level of pAkt, accompanied by a corresponding increase in pErk level when compared to patient-matched WBC. Finally, the current workflow successfully indicated the detectable pAkt and Akt signal difference in CTCs from two MBC patients: one with an Akt1 wild-type genotype, and the other harboring approximately 80% Akt1(E17K) mutated CTCs. The mutated CTCs revealed clearly elevated pAkt levels (1.8-fold), along with an even more strongly elevated total Akt (3.4-fold) when compared to the respective signals measured in wild-type CTCs. In conclusion, ZeptoCTC is a highly sensitive method for measuring the expression and phosphorylation of treatment-relevant proteins in key cancer-driving signaling pathways from true single cell samples.

molecular biology↗

bioRxiv2021

A platform of patient-derived microtumors identifies treatment response and therapeutic vulnerabilities of ovarian cancer

BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses. MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format. ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach. Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment.

cancer biology↗