Optical detection and virotherapy of live metastatic tumor cells in body fluids with vaccinia strains

Abstract

Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.

Figure 1. Detection and identification of live human CTCs in blood samples.

(A) VACV GLV-1h254 detected live CTCs in mice bearing human PC-3 prostate cancer xenografts. Infected CTCs were CK⁺ (B) or EpCAM⁺ (C). Live CTCs were also detected by GLV-1h254 in mice bearing human A549 non-small lung cancer xenografts (D). (E) The live CTCs in the patient BC1 were identified as TurboFP635⁺/EpCAM⁺ or CK⁺/CD45^−/DAPI⁺ cells. (F) The live CTCs detected in the patient BC5 were confirmed to express EpCAM, PR, or HER2/neu markers. (G) The live CTCs in the patient BC7 were confirmed as TurboFP635⁺/CK⁺/DAPI⁺ cells. (H) The live CTCs in the patient CC1 with metastatic colorectal cancer were identified as TurboFP635⁺/EpCAM⁺ or CK⁺/CD45^−/DAPI⁺ cells. (I) The live CTCs in the metastatic lung cancer patient LC1 were confirmed as TurboFP635⁺/EpCAM⁺/DAPI⁺ cells. (J) The VACV detected live CTCs in the metastatic melanoma patient MM1 were shown to express melanoma tumor markers Melan-A or microphthalmia-associated transcription factor.

Figure 2. Characterization of CTCs in blood samples detected with GLV-1h254.

(A) The CTCs in mice bearing human PC-3 prostate cancer xenografts showed high-level expression of CD44, ALDH1, vimentin and N-cadherin. (B) The CTCs from the human metastatic breast cancer patient BC1 showed the strong expression of CD44. (C) The live CTCs in the human metastatic breast cancer patient BC5 showed the strong expression of ALDH1.

Figure 3. Detection and identification of live tumor cells in a CSF sample.

(A) The live tumor cells in the CSF sample of a patient with metastatic breast cancer were identified as TurboFP635⁺/CK⁺/DAPI⁺ cells. (B) A live cancer cell cluster with heterogeneous expression of CK in the CSF sample was identified by VACV.

Figure 4. Prevention and therapy of CTCs in mice bearing human prostate cancer.

(A) GLV-1h68 early treatment prevented CTC formation in the majority of mice (7/8) while mice in the PBS group showed an increase in CTC numbers. (B) GLV-1h68 late treatment resulted in a dramatic decrease in CTC numbers while CTC numbers in the PBS group increased greatly, and then fluctuated over time. (C) Almost all CTCs detected in the GLV-1h68 late treatment group were GFP-positive (infected) at two weeks after treatment. (D) Primary tumors regressed after GLV-1h68 early or late treatment. (E) Both GLV-1h68 early and late treatments significantly prolonged mouse survival (p = 0.002, GLV-1h68 early vs. PBS; p = 0.006 GLV-1h68 late vs. PBS).

Figure 5. Therapy of cancer cells in the ascites of a cancer patient with peritoneal carcinomatosis.

Cell block sections of malignant ascitic cells were stained with haematoxylin and eosin (A-I, B-I) at three (A) and seven (B) days after virus treatment. EpCAM positive cells in the malignant ascites sections were detected using an anti-EpCAM antibody (A-II, B-II). GL-ONC1 infection of single cells was detected using an anti-VACV-A27L antibody (A-III, B-III). Scale bars represent 50 µm.