Monika Vishnoi

The isolation and characterization of CTC subsets related to breast cancer dormancy.

Uncovering CTCs phenotypes offer the promise to dissect their heterogeneity related to metastatic competence. CTC survival rates are highly variable and this can lead to many questions as yet unexplored properties of CTCs responsible for invasion and metastasis vs dormancy. We isolated CTC subsets from peripheral blood of patients diagnosed with or without breast cancer brain metastasis. CTC subsets were selected for EpCAM negativity but positivity for CD44+/CD24− stem cell signature; along with combinatorial expression of uPAR and int β1, two markers directly implicated in breast cancer dormancy mechanisms. CTC subsets were cultured in vitro generating 3D CTC tumorspheres which were interrogated for biomarker profiling and biological characteristics. We identified proliferative and invasive properties of 3D CTC tumorspheres distinctive upon uPAR/int β1 combinatorial expression. The molecular characterization of uPAR/int β1 CTC subsets may enhance abilities to prospectively identify patients who may be at high risk of developing BCBM.

Molecular characterization of breast cancer CTCs associated with brain metastasis

The enumeration of EpCAM-positive circulating tumor cells (CTCs) has allowed estimation of overall metastatic burden in breast cancer patients. However, a thorough understanding of CTCs associated with breast cancer brain metastasis (BCBM) is necessary for early identification and evaluation of treatment response to BCBM. Here we report that BCBM CTCs is enriched in a distinct sub-population of cells identifiable by their biomarker expression and mutational content. Deriving from a comprehensive analysis of CTC transcriptomes, we discovered a unique “circulating tumor cell gene signature” that is distinct from primary breast cancer tissues. Further dissection of the circulating tumor cell gene signature identified signaling pathways associated with BCBM CTCs that may have roles in potentiating BCBM. This study proposes CTC biomarkers and signaling pathways implicated in BCBM that may be used either as a screening tool for brain micro-metastasis detection or for making rational treatment decisions and monitoring therapeutic response in patients with BCBM.Characterization of CTCs derived from breast cancer patients with brain metastasis (BCBM) may allow for early diagnosis of brain metastasis and/or help for treatment choice and its efficacy. In this study, the authors identify a unique signature, based on patient-derived CTCs transcriptomes, for BCBM- CTCs that is different from primary tumors.

Circulating tumor cells from a 4-dimensional lung cancer model are resistant to cisplatin

OBJECTIVE To determine the effect of cisplatin on circulatory tumor cells (CTC) and tumor nodules in a four-dimensional (4D) lung cancer model. METHODS CTCs from the 4D model seeded with H1299, A549, or H460 and respective cells that were grown under two-dimensional conditions in a Petri dish were treated with 50 μM cisplatin for 24 and 48 hours and cell viability was determined. The lung nodules in the 4D model were then treated with different continuous or intermittent doses of cisplatin and the nodule size, the number of CTCs, and the level of matrix metalloproteinase (MMP) were determined. RESULTS Cisplatin led to a significant decrease in the viability of tumor cells grown under 2D conditions (P < .01) but not in CTCs from the 4D model after both 24 hours and 48 hours. Cisplatin led to regression of tumor nodules with both the continuous and intermittent treatments. Moreover, there was a significantly higher number of CTCs per tumor area (P < .05) and MMP-2 production per tumor area (P = .007) for all human lung cancer cell lines grown in the 4D model when treated with cisplatin. CONCLUSIONS The 4D lung cancer model allows for the isolation of CTCs that are resistant to cisplatin treatment. The model may allow us to better understand the biology of cisplatin resistance.

Targeting USP7 identifies a metastasis-competent state within bone marrow–resident melanoma CTCs

Systemic metastasis is the major cause of death from melanoma, the most lethal form of skin cancer. Although most patients with melanoma exhibit a substantial gap between onset of primary and metastatic tumors, signaling mechanisms implicated in the period of metastatic latency remain unclear. We hypothesized that melanoma circulating tumor cells (CTC) home to and reside in the bone marrow during the asymptomatic phase of disease progression. Using a strategy to deplete normal cell lineages (Lin-), we isolated CTC-enriched cell populations from the blood of patients with metastatic melanoma, verified by the presence of putative CTCs characterized by melanoma-specific biomarkers and upregulated gene transcripts involved in cell survival and prodevelopment functions. Implantation of Lin- population in NSG mice (CTC-derived xenografts, i.e., CDX), and subsequent transcriptomic analysis of ex vivo bone marrow-resident tumor cells (BMRTC) versus CTC identified protein ubiquitination as a significant regulatory pathway of BMRTC signaling. Selective inhibition of USP7, a key deubiquinating enzyme, arrested BMRTCs in bone marrow locales and decreased systemic micrometastasis. This study provides first-time evidence that the asymptomatic progression of metastatic melanoma can be recapitulated in vivo using patient-isolated CTCs. Furthermore, these results suggest that USP7 inhibitors warrant further investigation as a strategy to prevent progression to overt clinical metastasis.Significance: These findings provide insights into mechanism of melanoma recurrence and propose a novel approach to inhibit systematic metastatic disease by targeting bone marrow-resident tumor cells through pharmacological inhibition of USP7.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5349/F1.large.jpg Cancer Res; 78(18); 5349-62. ©2018 AACR.

Abstract 1530: Dissecting breast cancer dormant CTC phenotypes

Tumor relapse is a clinically relevant problem in breast cancer where patients are asymptomatic because disseminated cells appear to become dormant for periods longer than 20 years and are undetectable by current clinical tools. Uncovering phenotypes of circulating tumor cells (CTCs) - the “seeds” of intractable metastasis-offers the promise to dissect CTC heterogeneity in relation to metastatic competence, to predict biomarker assessment, and to significantly improve monitoring and treatment of cancer. However, little is known about CTC biology and how CTCs differ in their capacity to circulate while maintaining a metastatic potential. We hypothesized that EpCAM-negative breast cancer CTC subsets exist, and avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties. We collected peripheral blood of clinically diagnosed breast cancer patients with or without brain metastasis, and performed multiparametric flow cytometry to isolate EpCAM-negative CTC subsets with stem-cell properties (CD44+/CD24-), along with combinatorial expression of two neoplastic markers: urokinase plasminogen activator receptor (uPAR) and integrin beta1 (int β1). EpCAM-negative CTCs were further interrogated at a single-cell level employing DEPArray platform. Second, we were able to culture FACS-sorted CTC subsets, selected for six cell-surface expression markers (CD45-/EpCAM-negative/CD44+/CD24-/uPAR+/-/int β1+/-), as long-term in-vitro 3D CTC tumorspheres. Third, CTC subsets were interrogated for biomarker profiling and biological characteristics. We identified adhesive, proliferative and invasive properties of 3D CTC tumorspheres which were distinct per uPAR/int β1 combinatorial expression. Lastly, we performed next-generation whole-genome sequencing and mutation analyses to discover unique genomic signatures of uPAR/int β1 CTC subsets and verified as putative CTCs originally disseminated from primary breast tumor. Additional investigations are being pursued assessing the molecular and genomic characterization of uPAR/int β1 CTC subsets comprehensively. Clinical relevance of this research includes that this may enhance abilities to prospectively identify patients who may be at high-risk of developing breast cancer brain metastasis. Citation Format: Monika Vishnoi, Sirisha Peddibhotla, Wei Yin, Zhong Xue, Antonio T. Scamardo, Goldy C. George, David S. Hong, Dario Marchetti. Dissecting breast cancer dormant CTC phenotypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1530.

Abstract 3143: Deciphering mechanisms of circulating tumor cells in breast cancer dormancy

More than 67% of deaths in breast cancer patients occur after the initial 5-year survival period while residual disease can be dormant for periods longer than 20 years. Patients are asymptomatic because circulating tumor cells (CTCs) remain dormant and are undetectable by current clinical tools. Dormant CTCs may retain their long-term tumor-initiating (LTI) potential by adhering to their original genome, unlike rapidly cycling cancer cells that are known to have increased genomic instability. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to retain their LTI potential, ultimately causing disease relapse. We isolated and characterized EpCAM-negative breast cancer CTCs by mutiparametric flow cytometry and DEPArrayTM. Individually isolated breast cancer CTCs had a large proportion (>40%) of dormant (Ki67-/PCNA-) cells. Dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by the phosphorylation status of Serine139 on gamma H2AX. This observation was further validated in a panel of eight genetically distinct breast cancer cell lines. Second, to understand whether dormant cells are inherently more resistant to DSB, we induced DSB in breast cancer cells by UV radiation and bleomycin treatment, and measured residual DSB at regular intervals. Results showed that besides being more resistant to DSB de novo, dormant breast cancer cells were also more efficient in repairing their DNA. There are two distinct phases of DSB repair - early [within 2 hours of DSB using Non-Homologous End Joining (NHEJ) methods] and late [evident after 24 hours using Homologous Recombination (HR)]. Unlike proliferating (S-G2M) cells, dormant (G0) cells lack the sister chromatid and repair their DNA exclusively by NHEJ methods. Therefore, and third, we investigated key players of the NHEJ pathway and examined their roles in maintaining genomic integrity. We found that the human telomere-associated protein RIF1, a mediator of alternative NHEJ, was significantly up-regulated in a dormant CTC subset. Dormant sub-populations of breast cancer cells confirmed RIF1 foci formation in areas of DNA damage. Fourth, mis-sense mutation of RIF1 in CAMA-1 cells (ΔRIF1 E1598K) attenuated resistance of the dormant subset to UV and bleomycin treatment. Collectively, these findings suggest that RIF1 may play functional roles in maintaining the genomic integrity of dormant CTCs. Further investigations are being pursued to assess RIF1 contributions to retain CTC LTI potential leading to CTC-driven metastasis. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Antonio Scamardo, Goldy C. George, David S. Hong, Dario Marchetti. Deciphering mechanisms of circulating tumor cells in breast cancer dormancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3143.

Abstract 363: Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy

Uncovering phenotypes of patient-derived Circulating Tumor Cells (CTCs) offers the promise to dissect CTC heterogeneity in relation to metastatic competence, and to determine biomarkers of therapeutic utility for improved treatment. However, it is still unknown whether and how CTCs differ in their capacity to circulate while maintaining metastatic potential. Rates of CTC survival are highly variable, lasting less than few hours in some patients but in the order of decades in others. This can lead to many questions for yet unexplored mechanisms of CTCs responsible for dormancy, along with their properties and biomarker functionalities. We hypothesized that breast cancer CTC subsets possessing markers of pluripotency avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties; and that expression of urokinase plasminogen activator receptor (uPAR) and beta-1 integrin (β1int), two biomarkers known to be directly implicated in tumor cell dormancy, are relevant in controlling the recurrence of breast cancer brain metastasis (BCBM). First, we isolated CTC subsets not expressing the epithelial cell adhesion molecule (EpCAM-negative CTCs), and characterized these subsets using DEPArrayTM, a new CTC platform able to dissect CTC heterogeneity at a single-cell level, thus interrogating the smallest functional unit of cancer. We captured EpCAM-negative/CD45-/CD44+/CD24- breast cancer CTC subsets that possessed combinatorial uPAR and β1int expression using multiparametric flow cytometry. Second, CTC subsets grew in vitro and were further characterized by DEPArrayTM. Markers expression was confirmed by confocal microscopy with subsets possessing a specific breast cancer gene profiling. Third, EpCAM-negative CTC subsets (uPAR+/β1int+ and uPAR-/β1int-) were interrogated for human embryonic stem cell markers by RT2 PCR arrays. Gene expression profiling was consistently distinct among uPAR+/β1int+ vs. uPAR-/β1int- CTC subsets and dependent upon patients’ BCBM status: expression of genes implicated in cell cycle progression (e.g., CDK42, CDK1), angiogenesis (e.g., FGF-2), and pluripotency (e.g., KLF4) was >30-fold higher than controls. Third, CTC subsets gene patterns isolated from patients with BCBM possessed RT2 profiles that were strikingly distinct from ones derived from patients with no BCBM. Of note, gene expression for RIF-1, a protein that counteracts actions of the breast cancer suppressor BRCA1, was highest (>50-fold) with distinct RIF-1 nuclear patterns in BCBM CTC subsets. In summary, we have linked EpCAM-negative uPAR/β1int CTC subsets and their properties to clinical BCBM; and will assess the therapeutic inhibition of uPAR/β1int CTC biomarkers on BCBM development and its timing. Deciphering the relevance of uPAR/β1int as key CTC biomarkers of dormancy vs. metastatic competence will elucidate CTC mechanisms responsible for BCBM onset. Citation Format: Sirisha Peddibhotla, Monika Vishnoi, Wei Yin, Yizhen Chen, Antonio Scamardo, David Hong, Dario Marchetti. Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 363. doi:10.1158/1538-7445.AM2015-363