Longitudinal Monitoring of Intra-Tumoural Heterogeneity Using Optical Barcoding of Patient-Derived Colorectal Tumour Models.

Geno- and phenotypic heterogeneity amongst cancer cell subpopulations are established drivers of treatment resistance and tumour recurrence. However, due to the technical difficulty associated with studying such intra-tumoural heterogeneity, this phenomenon is seldom interrogated in conventional cell culture models. Here, we employ a fluorescent lineage technique termed "optical barcoding" (OBC) to perform simultaneous longitudinal tracking of spatio-temporal fate in 64 patient-derived colorectal cancer subclones. To do so, patient-derived cancer cell lines and organoids were labelled with discrete combinations of reporter constructs, stably integrated into the genome and thus passed on from the founder cell to all its clonal descendants. This strategy enables the longitudinal monitoring of individual cell lineages based upon their unique optical barcodes. By designing a novel panel of six fluorescent proteins, the maximum theoretical subpopulation resolution of 64 discriminable subpopulations was achieved, greatly improving throughput compared with previous studies. We demonstrate that all subpopulations can be purified from complex clonal mixtures via flow cytometry, permitting the downstream isolation and analysis of any lineages of interest. Moreover, we outline an optimized imaging protocol that can be used to image optical barcodes in real-time, allowing for clonal dynamics to be resolved in live cells. In contrast with the limited intra-tumour heterogeneity observed in conventional 2D cell lines, the OBC technique was successfully used to quantify dynamic clonal expansions and contractions in 3D patient-derived organoids, which were previously demonstrated to better recapitulate the heterogeneity of their parental tumour material. In summary, we present OBC as a user-friendly, inexpensive, and high-throughput technique for monitoring intra-tumoural heterogeneity in in vitro cell culture models.

CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM.

Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches-immune detection coupled with magnetic beads and fluorescence-activated cell sorting-were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.

CSK-homologous kinase (CHK/MATK) is a potential colorectal cancer tumour suppressor gene epigenetically silenced by promoter methylation.

Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

Laminin 521 enhances self-renewal via STAT3 activation and promotes tumor progression in colorectal cancer.

Remodeling of basement membrane proteins contributes to tumor progression towards the metastatic stage. One of these proteins, laminin 521 (LN521), sustains embryonic and induced pluripotent stem cell self-renewal, but its putative role in cancer is poorly described. In the present study we found that LN521 promotes colorectal cancer (CRC) cell self-renewal and invasion. siRNA-mediated knockdown of endogenously-produced laminin alpha 5, as well as treatment with neutralizing antibodies against integrin α3ß1 and α6ß1, were able to reverse the effect of LN521 on self-renewal. Exposure of CRC cells to LN521 enhanced STAT3 phosphorylation, and incubation with STAT3 inhibitors Napabucasin and Stattic was sufficient to block the LN521-driven self-renewal increase. Robust expression of laminin alpha 5 was detected in 7/10 liver metastases tissue sections collected from CRC patients as well as in mouse liver metastasis xenografts, in most cases within areas expressing metastasis cancer stem cell markers such as c-KIT and CD44v6. Finally, retrospective analysis of multiple CRC datasets highlighted the significant association between high LN521 mRNA expression and poor clinical outcome in colorectal cancer patients. Collectively our results indicate that high Laminin 521 expression is a frequent feature of metastatic dissemination in CRC and that it promotes cell invasion and self-renewal, the latter through engagement of integrin isoforms and activation of STAT3 signaling.

Compromized DNA repair as a basis for identification of cancer radiotherapy patients with extreme radiosensitivity.

A small percentage of cancer radiotherapy patients develop abnormally severe side effects as a consequence of intrinsic radiosensitivity. We analysed the γ-H2AX response to ex-vivo irradiation of peripheral blood lymphocytes (PBL) and plucked eyebrow hair follicles from 16 patients who developed severe late radiation toxicity following radiotherapy, and 12 matched control patients. Longer retention of the γ-H2AX signal and lower colocalization efficiency of repair factors in over-responding patients confirmed that DNA repair in these individuals was compromised. Five of the radiosensitive patients harboured LoF mutations in DNA repair genes. An extensive range of quantitative parameters of the γ-H2AX response were studied with the objective to establish a predictor for radiosensitivity status. The most powerful predictor was the combination of the fraction of the unrepairable component of γ-H2AX foci and repair rate in PBL, both derived from non-linear regression analysis of foci repair kinetics. We introduce a visual representation of radiosensitivity status that allocates a position for each patient on a two-dimensional "radiosensitivity map". This analytical approach provides the basis for larger prospective studies to further refine the algorithm, ultimately to triage capability.

Radiotherapy for Non-Small Cell Lung Cancer Induces DNA Damage Response in Both Irradiated and Out-of-field Normal Tissues.

PURPOSE: To study the response of irradiated and out-of-field normal tissues during localized curative intent radiotherapy. EXPERIMENTAL DESIGN: Sixteen patients with non-small cell lung carcinoma (NSCLC) received 60 Gy in 30 fractions of definitive thoracic radiotherapy with or without concurrent chemotherapy. Peripheral blood lymphocytes (PBL) and eyebrow hairs were sampled prior, during, and after radiotherapy. Clinical variables of radiotherapy dose/volume, patient age, and use of chemoradiotherapy were tested for association with γ-H2AX foci, a biomarker of DNA damage that underlies cellular response to irradiation. RESULTS: Radiotherapy induced an elevation of γ-H2AX foci in PBL, representing normal tissues in the irradiated volume, 1 hour after fraction one. The changes correlated directly with mean lung dose and inversely with age. γ-H2AX foci numbers returned to near baseline values in 24 hours and were not significantly different from controls at 4 weeks during radiotherapy or 12 weeks after treatment completion. In contrast, unirradiated hair follicles, a surrogate model for out-of-field normal tissues, exhibited delayed "abscopal" DNA damage response. γ-H2AX foci significantly increased at 24 hours post-fraction one and remained elevated during treatment, in a dose-independent manner. This observed abscopal effect was associated with changes in plasma levels of MDC/CCL22 and MIP-1α/CCL3 cytokines. No concordant changes in size and concentration of circulating plasma exosomes were observed. CONCLUSIONS: Both localized thoracic radiotherapy and chemoradiotherapy induce pronounced systemic DNA damage in normal tissues. Individual assessment of biologic response to dose delivered during radiotherapy may allow for therapeutic personalization for patients with NSCLC. Clin Cancer Res; 22(19); 4817-26. ©2016 AACRSee related commentary by Verma and Lin, p. 4763.

Evaluation of Severe Combined Immunodeficiency and Combined Immunodeficiency Pediatric Patients on the Basis of Cellular Radiosensitivity.

Pediatric patients with severe or nonsevere combined immunodeficiency have increased susceptibility to severe, life-threatening infections and, without hematopoietic stem cell transplantation, may fail to thrive. A subset of these patients have the radiosensitive (RS) phenotype, which may necessitate conditioning before hematopoietic stem cell transplantation, and this conditioning includes radiomimetic drugs, which may significantly affect treatment response. To provide statistical criteria for classifying cellular response to ionizing radiation as the measure of functional RS screening, we analyzed the repair capacity and survival of ex vivo irradiated primary skin fibroblasts from five dysmorphic and/or developmentally delayed pediatric patients with severe combined immunodeficiency and combined immunodeficiency. We developed a mathematical framework for the analysis of γ histone 2A isoform X foci kinetics to quantitate DNA-repair capacity, thus establishing crucial criteria for identifying RS. The results, presented in a diagram showing each patient as a point in a 2D RS map, were in agreement with findings from the assessment of cellular RS by clonogenic survival and from the genetic analysis of factors involved in the nonhomologous end-joining repair pathway. We provide recommendations for incorporating into clinical practice the functional assays and genetic analysis used for establishing RS status before conditioning. This knowledge would enable the selection of the most appropriate treatment regimen, reducing the risk for severe therapy-related adverse effects.

A pattern of early radiation-induced inflammatory cytokine expression is associated with lung toxicity in patients with non-small cell lung cancer.

PURPOSE: Lung inflammation leading to pulmonary toxicity after radiotherapy (RT) can occur in patients with non-small cell lung cancer (NSCLC). We investigated the kinetics of RT induced plasma inflammatory cytokines in these patients in order to identify clinical predictors of toxicity. EXPERIMENTAL DESIGN: In 12 NSCLC patients, RT to 60 Gy (30 fractions over 6 weeks) was delivered; 6 received concurrent chemoradiation (chemoRT) and 6 received RT alone. Blood samples were taken before therapy, at 1 and 24 hours after delivery of the 1st fraction, 4 weeks into RT, and 12 weeks after completion of treatment, for analysis of a panel of 22 plasma cytokines. The severity of respiratory toxicities were recorded using common terminology criteria for adverse events (CTCAE) v4.0. RESULTS: Twelve cytokines were detected in response to RT, of which ten demonstrated significant temporal changes in plasma concentration. For Eotaxin, IL-33, IL-6, MDC, MIP-1α and VEGF, plasma concentrations were dependent upon treatment group (chemoRT vs RT alone, all p-values <0.05), whilst concentrations of MCP-1, IP-10, MCP-3, MIP-1ß, TIMP-1 and TNF-α were not. Mean lung radiation dose correlated with a reduction at 1 hour in plasma levels of IP-10 (r2 = 0.858, p<0.01), MCP-1 (r2 = 0.653, p<0.01), MCP-3 (r2 = 0.721, p<0.01), and IL-6 (r2 = 0.531, p = 0.02). Patients who sustained pulmonary toxicity demonstrated significantly different levels of IP-10 and MCP-1 at 1 hour, and Eotaxin, IL-6 and TIMP-1 concentration at 24 hours (all p-values <0.05) when compared to patients without respiratory toxicity. CONCLUSIONS: Inflammatory cytokines were induced in NSCLC patients during and after RT. Early changes in levels of IP-10, MCP-1, Eotaxin, IL-6 and TIMP-1 were associated with higher grade toxicity. Measurement of cytokine concentrations during RT could help predict lung toxicity and lead to new therapeutic strategies.

Mobilization of viable tumor cells into the circulation during radiation therapy.

PURPOSE: To determine whether radiation therapy (RT) could mobilize viable tumor cells into the circulation of non-small cell lung cancer (NSCLC) patients. METHODS AND MATERIALS: We enumerated circulating tumor cells (CTCs) by fluorescence microscopy of blood samples immunostained with conventional CTC markers. We measured their DNA damage levels using γ-H2AX, a biomarker for radiation-induced DNA double-strand breaks, either by fluorescence-activated cell sorting or by immunofluorescence microscopy. RESULTS: Twenty-seven RT-treated NSCLC patients had blood samples analyzed by 1 or more methods. We identified increased CTC numbers after commencement of RT in 7 of 9 patients treated with palliative RT, and in 4 of 8 patients treated with curative-intent RT. Circulating tumor cells were also identified, singly and in clumps in large numbers, during RT by cytopathologic examination (in all 5 cases studied). Elevated γ-H2AX signal in post-RT blood samples signified the presence of CTCs derived from irradiated tumors. Blood taken after the commencement of RT contained tumor cells that proliferated extensively in vitro (in all 6 cases studied). Circulating tumor cells formed γ-H2AX foci in response to ex vivo irradiation, providing further evidence of their viability. CONCLUSIONS: Our findings provide a rationale for the development of strategies to reduce the concentration of viable CTCs by modulating RT fractionation or by coadministering systemic therapies.

Tumour targeting of Auger emitters using DNA ligands conjugated to octreotate.

PURPOSE: The objective of the study was to conjugate the DNA binding ligand para-[(125)I]-iodoHoechst to octreotate, and to explore the tumour targeting potential of this conjugate in the octreotate-somatostatin receptor system. METHODS: We synthesized a Hoechst analogue containing a tri-butylstannyl group in the para position of phenyl ring, conjugated it to the N-terminal amino group of octreotate and prepared (125)I-labelled conjugate by iododestannylation. We used the somatostatin receptor (SSTR2) over-expressing cell line A427-7 derived from its parent A427 human non-small cell lung carcinoma cell line to investigate SSTR2 affinity and receptor-mediated internalisation of the conjugate, and the mouse A427-7 tumour xenograft model for in vivo biodistribution studies of the radiolabelled conjugate. RESULTS: A method was developed for convenient preparation of high specific activity radioiodinated conjugate which retains affinity for somatostatin receptors and is internalised into A427-7 SSTR2 over-expressing cells via a receptor-mediated mechanism. The conjugate accumulates in mouse A427-7 tumour xenografts following intravenous administration. CONCLUSIONS: A dual targeting strategy for Auger endoradiotherapy, in which a DNA ligand is used to target the Auger decay to DNA, in conjunction with receptor-mediated targeting to specific receptors, using a labelled DNA ligand/peptide conjugate, has been demonstrated for the octreotate-somatostatin receptor system.