Methionine addiction is a fundamental and universal hallmark of cancer (1-9) termed the Hoffman effect (10). Numerous pre-clinical studies, both in vitro and in mouse models of cancer (11), have shown that all tested cancers are methionine addicted and respond to methionine restriction with recombinant methioninase (rMETase). Recently, clinical case studies targeting methionine addiction with oral rMETase (o-rMETase) have shown potential for efficacy against recalcitrant cancer (12-17). It is therefore important to identify patients who would be appropriate candidates for o-rMETase treatment.

We developed the histoculture drug response assay (HDRA) almost forty years ago (18-22). The HDRA uses 3-dimensional cultures of tumor fragments on Gelfoam substrates. There is a high correlation of HDRA results and chemotherapy outcome in the clinic, including survival (20-22).

In the present study, we utilized the HDRA to determine sensitivity of highly recalcitrant peritoneal-metastatic cancer to rMETase. We propose that rMETase sensitivity in the HDRA is a potential biomarker to indicate methionine-restriction therapy in the clinic.

Patients. In the present study, we focused on highly recalcitrant peritoneal-metastatic cancers. Tissue samples were collected from 27 colorectal cancer cases, an appendix cancer case, six pancreatic cancer cases, eight ovarian cancer cases, and seven pseudomyxoma patients who underwent surgery at the Kishiwada Tokushukai Hospital in Japan. The experimental use of the chemosensitivity test was approved by the Institutional Research Committee in Kishiwada Tokushukai Hospital. HDRA testing was approved by the Institutional Review Board for the University of Fukui Hospital. All patients were informed of the nature of this study, and written informed consent was obtained.

HDRA. The HDRA was performed as an in vitro assay for assessing rMETase sensitivity, as described in prior studies (18-22). The collagen sponge gels (Gelfoam^®) used in this study were obtained from Pfizer Japan Inc. (Tokyo, Japan). Gelfoam was manufactured using pig skin as the primary raw material. The cancerous portions of the specimens were cut into fragments weighing approximately 10 mg. These fragments were placed on the surface of Gelfoam in 24-well microplates. The plates were incubated for seven days at 37˚C with a humidity level of 100% and atmospheric composition of 95% air and 5% CO₂ in the presence of rMETase dissolved in RPMI 1640 medium containing 20% fetal calf serum. rMETase was tested at 2.5 U/ml, 10 U/ml, and 20 U/ml. The incubation temperature was maintained at 37˚C. Following specimen histoculture, a solution consisting of 100 μl Hank’s balanced salt solution containing 0.1 mg/ml type I collagenase (Sigma, St. Louis, MO, USA) and 100 μl of a solution of 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dissolved in phosphate-buffered saline (9.6 mg/ml) was added to each culture well. The specimens were then incubated for an additional 16 h. After an extraction process using dimethylsulfoxide, the absorbance of the solution in each well was measured at a wavelength of 540 nm (with control at 630 nm) using a microplate reader (Spectra Max M5; Molecular Devices LLC, San Jose, CA, USA). The absorbance per mg of cultivated tumor tissue was determined by averaging the absorbance values (OD) obtained from three separate culture wells. The weight of the tumor tissue was measured before initiating the culture. The calculation of the inhibition rate was performed using the following formula:

Inhibition rate (%)=(1 - mean OD/well of treated

well/mean OD/well of control well) ×100

rMETase production and formulation. Escherichia coli that had been transformed with the methioninase gene derived from Pseudomonas putida was fermented in order to produce rMETase. The purification steps comprised heating at 60˚C, followed by polyethylene glycol precipitation and diethylaminoethyl-sepharose fast-flow non-exchange chromatography. rMETase was dissolved in a 5 mg/ml solution of pyridoxal 5’-phosphate (PLP) in normal saline (23,24).

Statistics. Statistical analyses were performed using GraphPad Prism 9.4.0 (GraphPad Software, Inc., San Diego, CA, USA). The data are presented as the mean and standard deviation. The significance threshold was less than or equal to 0.05.

Sensitivity of peritoneal-metastatic colorectal cancer and appendix cancer to rMETase in the HDRA. Peritoneal metastatic colorectal and appendix cancer (N=27) were sensitive to rMETase in the HDRA with an inhibition rate ranging from 34.9 % to 40.6 % (Figure 1). There was no dose-response effect as 2.5 units rMETase was almost as effective as 10 and 20 units/ml rMETase.

Sensitivity of peritoneal-metastatic pancreatic cancer to rMETase in the HDRA. Peritoneal-metastatic pancreatic cancer (N=6) was sensitive to rMETase in the HDRA with an inhibition rate ranging from 29.0 to 38.3% with moderate dose-response to rMETase from 2.5 to 20 units/ml (Figure 2).

Sensitivity of peritoneal-metastatic ovarian cancer to rMETase in the HDRA. Peritoneal-metastatic ovarian cancer (N=8) was sensitive to rMETase in the HDRA with an inhibition rate ranging from 22.8 to 33.9 % for 2.5 units/ml to 20 units/ml rMETase (Figure 3).

Sensitivity of peritoneal-metastatic pseudomyxoma to rMETase in the HDRA. Peritoneal-metastatic pseudomyxoma (N=7) was sensitive to rMETase in the HDRA with an inhibition rate ranging from 36.7 to 42.0 %. Dose response was not observed for rMETase from 2.5 to 20 units/ml (Figure 4).

Methionine addiction is a universal hallmark of cancer (1-9). Therefore, it is a prime therapeutic target, especially for highly-recalcitrant cancer such as peritoneal-metastatic cancer, described in the present report. The present study determined the sensitivity of peritoneal-metastatic cancer to rMETase in the HDRA. The HDRA allows tissues to take their natural shape in 3-dimensions on a flexible in vivo-like substrate, enabling clinically-accurate drug sensitivity results to be obtained (18-22).

Colorectal cancer, appendix cancer, and pseudomyxoma tended to be more sensitive to rMETase than ovarian and pancreatic cancer in the HDRA. In pancreatic and ovarian cancer, there was moderate dose-dependency of rMETase in the HDRA.

When cancer has spread to the peritoneum, the disease is highly recalcitrant and most often lethal. The present study shows for the first time the response of peritoneal-metastatic cancer to rMETase. Since the HDRA has previously shown a high correlation with clinical results (20,22), the present results suggest that rMETase has clinical potential as therapy of peritoneal-metastatic cancer. The present study suggests that sensitivity of a cancer to rMETase in the HDRA could be a functional biomarker for sensitivity to methionine-restriction therapy in the clinic.

The Authors declare no competing interests regarding this work.

MS performed experiments. YK, and RMH wrote the article. QH provided methioninase. CH and TT gave critical suggestions.

This paper is dedicated to the memory of A. R. Moossa, MD, Sun Lee, MD, Gordon H Sato, PhD, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Jack Geller, MD, Joseph R Bertino, MD, and J.A.R. Mead, PhD. The Robert M Hoffman Foundation for Cancer Research provided funds for this study.