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A colon cancer growth inhibitor partially purified from the isolated cell wall membrane fraction of Chlorella sorokiniana, here referred to as Chlorella membrane factor (CMF), was evaluated for its antitumor and immunomodulatory effects in cell culture and in a colon carcinoma mouse model. The CMF treatment dose- and time-dependently inhibited colon carcinoma cell growth in 2-dimensional cultures. Treatment with CMF also significantly inhibited the growth of colon carcinoma spheroids in 3-dimensional cell culture in coculture with T lymphocytes. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMF (10 or 30 mg dry weight/kg body weight, every other day) dose-dependently and significantly attenuated the growth of tumor nodules via induction of tumor cell apoptosis. Evaluation of immune cell populations in ascites showed that CMF treatment tended to increase T lymphocytes but lower granulocyte populations. The present study suggests that the cell wall membrane fraction of Chlorella sorokiniana contains a bioactive material that inhibits colon carcinoma growth via direct cell growth inhibition and stimulation of host antitumor immunity. Hence, it is suggested that the Chlorella cell wall membrane extract or a bioactive substance in the extract is an attractive complementary medicine for cancer therapy.

Beta glucans are known to have immunomodulatory effects that mediated by a variety of mechanisms. In this article, we describe experiments and simulations suggesting that beta-1,3 glucans may promote activation of T cells by a previously unknown mechanism. First, we find that treatment of a T lymphoblast cell line with beta-1,3 oligoglucan significantly increases mRNA levels of T cell activation-associated cytokines, especially in the presence of the agonistic anti-CD3 antibody. This immunostimulatory activity was observed in the absence of dectin-1, a known receptor for beta-1,3 glucans. To clarify the molecular mechanism underlying this activity, we performed a series of molecular dynamics simulations and free-energy calculations to explore the interaction of beta-1,3 oligoglucans with potential immune receptors. While the simulations reveal little association between beta-1,3 oligoglucan and the immune receptor CD3, we find that beta-1,3 oligoglucans bind to CD28 near the region identified as the binding site for its natural ligands CD80 and CD86. Using a rigorous absolute binding free-energy technique, we calculate a dissociation constant in the low millimolar range for binding of 8-mer beta-1,3 oligoglucan to this site on CD28. The simulations show this binding to be specific, as no such association is computed for alpha-1,4 oligoglucan. This study suggests that beta-1,3 glucans bind to CD28 and may stimulate T cell activation collaboratively with T cell receptor activation, thereby stimulating immune function.

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Phytochemicals have been developed into treatments against many diseases, including various cancers. Euglena is a genus of single-cell flagellate eukaryotes having features of both animals and plants. These organisms, which are found in both fresh and salt water, have been used as a dietary supplement for their rich nutrient content. Although recent studies suggest that Euglena extracts have anti-tumor properties, the mechanism is yet to be clarified. Accordingly, the anti-tumor and immunomodulatory effects of partially purified water extract from Euglena gracilis (EWE) was evaluated in cell culture and a mouse orthotopic lung carcinoma allograft model. The EWE was prepared by a PBS extraction at 37°C for 30 min, centrifugation at 2,000 g for 20 min, and filtration through a membrane filter (0.22 μm pore size). Primary components of this extract are water soluble materials and exclude mature paramylons, intact chloroplasts, and oil droplets. In two-dimensional cell culture, the EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells (A549 and H1299) in a dose (1-100 μg/ml) and time (24-72 h)-dependent manner. In contrast, the growth of mouse bone marrow cells (BMCs), but not mouse splenocytes (SPLs) were dose (1-100 μg/ml) and time (24-72 h)-dependently stimulated by the treatment with EWE. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated by EWE treatment. Flow cytometry revealed that the EWE treatment attenuated granulocytic myeloid-derived suppressor cells in BMCs. In a mouse LLC orthotopic allograft model, pretreatment with EWE (100-200 mg/kg/day, via drinking water) three weeks prior to LLC cell inoculation significantly attenuated the growth of LLC tumors in the lungs of immunocompetent syngeneic mice. However, this attenuation was not seen for EWE treatment initiated after LLC cell inoculation. The tumor growth attenuation by EWE pretreatment coincided with a significant decrease of myeloid-derived cells, primarily neutrophils. EWE pretreatment appears to inhibit lung carcinoma growth mainly by stimulating host anti-tumor immunity via an attenuation of the host myeloid-derived immune suppressor cells. Hence, the present study suggests that the partially purified extract derived from Euglena gracilis contains bioactive materials that prevent lung carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT and JC), a Kansas State University College of Veterinary Medicine SMILE award (MT and JC), and NIH grant P20 RR017686 (MT). Citation Format: Susumu Ishiguro, Deepa Upreti, Nicole Robben, Page Cote, Riley Burghart, Damilola Ogun, Tran Le, Jennifer Delzeit, Jayson Carswell, Arashi Nakashima, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Water extract from Euglena gracilis prevents lung carcinoma growth in mice via attenuation of myeloid-derived suppressor cells and granulocytes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3459.