STUDYING THE EFFECTS OF AHCC ON COLORECTAL CANCER AND THE
UNDERLYING MECHANISM
by
GABRIEL D. MOSS
Submitted in partial fulfillment of the requirements for the degree of Master of Science
Department of Anatomy
CASE WESTERN RESERVE UNIVERSITY
May 2021
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Gabriel D. Moss
candidate for the degree of (Master of Science)
Committee Chair
Darin A. Croft
Committee Members
Andrew R. Crofton, PhD
Richard T. Lee, MD
Date of Defense
(3/24/2021)
*We also certify that written approval has been obtained for any proprietary material contained therein.
2 TABLE OF CONTENTS
TABLE OF CONTENTS…………………………………………………………..……3
LIST OF FIGURES…………………………………………………………………...…4
ACKNOWLEDGMENTS………………………………………………………………..5
ABSTRACT………………………………………………………………………………6
INTRODUCTION………………………………………………………………………..8
METHODS………………………………………………………..…………………….16
Mice……………………………………………………………………………..16
AHCC Preparation……………………………………………………………..16
Aim 1: Dosing Study…………………………………………………………..16
Aim 2: Long-Term Study……………………………………………………...17
Aim 3: Mechanism……………………………………………………………..18
RESULTS…………………………………………...………………………………….20
Aim 1: Dosing Study…………………………………………………………..20
Aim 2: Long-Term Study………………………………………………………20
Aim 3: Mechanism……………………………………………………,,………24
DISCUSSION…………………………………………………………………….…….25
CONCLUSION…………………………………………………………………...…….29
REFERENCES…………………………………………………………………………30
3 LIST OF FIGURES
Figure 1- Structure of AHCC………………………………………………………….10
Figure 2- Five-year overall survival curve comparing patients receiving immunotherapy (PSK) and chemotherapy (control)………………….………...….11
Figure 3- Kaplan-Meier estimates of the no recurrence rate and overall survival of HCC patients after hepatic resection…………………………………………..……12
Figure 4- Clinical responses to Pembrolizumab treatment in colorectal cancer…………………………………………………………………………………...13
Figure 5- AHCC treatment in vitro…………………………………..……………….14
Figure 6- AHCC treatment in vivo……………………………………………………15
Figure 7- AHCC treatment in NSG mice…………………………………………….15
Figure 8- FACS Analysis on Tumors………………………………………...………19
Figure 9- AHCC Dosing Study…………………………………………………….…20
Figure 10- AHCC Long Term Study, Change in Volume……………………….....22
Figure 11- AHCC Long-Term Study, Percent Change from Initial Tumor Size at Day 10…………………………………………………………………………………..23
Figure 12- AHCC Long-Term Study Survival Curve………...……………………..24
Figure 13- FACS Analysis on Peripheral Blood…………………………………….25
4 ACKNOWLEDGMENTS
I would like to thank my mentor Dr. Richard Lee for his guidance, insight, and dedication to my project. I would also like to thank Saada Eid for her daily help in the laboratory, teaching me techniques, and helping troubleshoot. Additionally, I would like to express my gratitude to Dr. Alex Huang for providing his expertise regarding immunology and potential experiments. Finally, I would like to thank the other members of my thesis committee, Dr. Darin Croft and Dr. Andrew
Crofton for providing their time and insight on how to strengthen my study.
5 Studying the Effects of AHCC On Colorectal Cancer and the Underlying
Mechanism
Abstract
By
GABRIEL D. MOSS
Purpose: This study sought to determine the anticancer effects of amino hexose correlated compound (AHCC) on colorectal cancer (CRC) and the underlying mechanism.
Background: CRC is the third most common cancer type and is the second leading cause of cancer deaths, with a 5-year survival rate of only 10-12% in patients with Stage IV CRC. Mushroom extracts contain immunomodulatory glucans that are known to have anti-cancer properties.
Methods: CT26, a murine derived CRC line, was injected into female Balb/c mice to determine the optimal dosage and long-term effects, 10 days later AHCC was given daily via oral gavage to determine the optimal dose. To determine the long- term effects, 120mg of AHCC was given 10 days after CT26 injection. Arm A received no AHCC (control), Arm B received AHCC daily, Arm C received AHCC through day 14 and then permanently stopped treatment, and Arm D received
AHCC through day 14, temporarily stopped until day 21, when it was
6 reintroduced. FACs staining was used to analyze peripheral blood 24 and 72 hours after a single AHCC treatment, specifically looking at monocytes.
Results: We found that the optimal dose of AHCC for Balb/c mice is 120 mg, which reduced tumor growth by 58.3% (p<0.05) compared to controls.
Additionally, we found that stopping and restarting treatment appears to improve its anticancer effects by 29-49% on days 14-24 post-injection (p<0.05). Finally, we found that AHCC appears to cause a significant decrease in MHCII+CD11B+ and MHCII+CD11C+ cell populations by 55-58% at 24 hours (p<0.05).
Conclusion: These results indicate that AHCC has immunomodulatory anticancer effects in a CRC mouse model and these findings could potentially translate into new therapies and new AHCC dosing schedules in advanced cancer patients.
Mechanistically, AHCC may be immediately mobilizing macrophages, monocytes, and dendritic cells from circulation into the periphery, potentially to the tumor and/or shifting the phenotype of a subset of dendritic cells or macrophages. This effect dissipated by 72 hours as immune cell numbers normalized.
7 INTRODUCTION
Colorectal cancer (CRC) is the third most prevalent cancer globally and in the United States, with approximately 145,000 new cases and 53,000 deaths in the United States in 2020 (Cancer Fast Facts and Figures, 2020). It is the second leading cause of cancer fatalities, accounting for 9% of all cancer deaths worldwide (Bastos et al., 2010). Generally, those diagnosed with Stage II with high-risk features and all Stage III cancers are treated with surgery and adjuvant chemotherapy and have a five-year survival rate of 50-90% (Overman et al.,
2017). Stage IV cancers are treated with chemotherapy, though are generally incurable, with a five-year survival rate of only 10% with current therapies (Wang et al., 2020).
Immunotherapies stimulate the immune system to target the cancer and are becoming an increasingly popular area of study and treatment, specifically cell program death protein 1 (PD-1) inhibitors (Overman et al., 2017). PD-1 is an immune checkpoint receptor on activated T cells that leads to immunosuppression when it binds to its ligand (PDL-1) on a target cell. Thus, by suppressing this interaction via a PD-1/L-1 inhibitor, immune responses can be enhanced by allowing T cells to continue to activate other immune cells, regulate the immune response, and destroy affected cells (Topalian et al., 2012).
Currently, six PD-1/L1 inhibitors are approved for various cancers (Overman et al., 2017), including pembrolizumab and nivolumab, which were approved as a second line treatment for DNA mismatch repair deficient/microsatellite instability
8 high metastatic (MSI-H) CRC in 2017 (Diaz et al., 2017; Overman et al., 2017).
Additionally, in 2020, pembrolizumab was approved as a first line treatment for this metastatic CRC tumor subtype (Andre et al., 2020). While MSI-H CRC tumors respond to immunotherapy drugs such as pembrolizumab and nivolumab at a rate of roughly 50%, MSI-H tumors only account for approximately 5% of advanced CRC (Overman et al, 2017; Le et al., 2017). Conversely, MSI-low
(MSI-L) CRC account for the majority of advanced CRCs yet have a response rate of less than 5% to immunotherapies (Battaglin et al., 2018), thus a new treatment strategy is needed for metastatic MSI-L CRC.
Natural products have long been a popular source for drug discovery, accounting for 41% of anticancer drugs approved between 1940 and 2010
(Newman et al., 2012). Mushrooms specifically have been used in traditional
Chinese medicine for centuries and have been shown to contain glucans, glucose derived polysaccharides, which have immunomodulatory effects (Novak et al., 2008). Several groups have been able to show the anticancer immune mediated effects of these glucans, such as activating immune cells including macrophages and natural killer (NK) cells (Ayeka et al., 2018; Lemieszek et al.,
2019; Wang et al., 2015). Similar mushroom compounds, such as polysaccharide
K (PSK), have been shown to reduce recurrence and improve survival among
CRC patients in randomized controlled clinical trials (Figure 2) (Oba et al., 2007;
Sakamoto et al., 2006). Amino hexose correlated compound (AHCC) is a mushroom extract derived from Lentinula edodes, a fungus that is commonly known as the shiitake mushroom, and mainly contains alpha (1-4) glucans
9 (Figure 1) (Oba et al., 2007; Sakamoto et al., 2006; Olamigoke et al., 2015). In previous cohort studies with patients with hepatic cancer, AHCC has been shown to decrease recurrence and increase overall survival (Cowawintaweewat et al.,
2006), and increase overall survival (Figure 3) (Matsui et al., 2002), though no work has been done with randomized placebo-controlled trials.
While the mechanism by which AHCC promotes its anti-cancer effects are not clear, one proposed mechanism involves the modulation of toll-like receptor 2
(TLR2) and TLR4 to create and maintain immune homeostasis and may also have a positive effect on intestinal epithelial cells, though few studies have examined AHCC and its mechanism in CRC (Daddaoua et al., 2013; Mallet et al.,
2016).
Figure 1: Structure of AHCC (Olamigoke et al., 2015).
10
Figure 2: Five-year overall survival curve comparing patients receiving immunotherapy (PSK) and chemotherapy (control) (Sakamoto et al., 2006).
11
Figure 3: Kaplan-Meier estimates of the no recurrence rate and overall survival of HCC patients after hepatic resection. The thick line indicates survival in the AHCC group, and the thin line represents the control group. Overall survival. There was also a significant difference between the two groups (P=0.0032) (Matsui et al., 2002).
In previous studies, Overman et al. (2017) and Le et al. (2017) showed that MSI-H CRCs have a response rate of 50% to PD-1 inhibitors, while Battaglin et al. show that the more common MSI-L CRC only respond to PD-1 inhibitors at a rate of less than 5% (Figure 4), thus, an alternative strategy is needed to treat metastatic CRC, specifically MSI-L CRC. One method has been to activate the innate immune system, which when combined with a PD-1 inhibitor has been shown to enhance the anti-tumor effects in animal models (Dong et al., 2019;
Kather et al., 2019). This combinatory strategy is currently undergoing clinical trials in CRC patients with imprime β(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-
12 β(1,3)-D-glucopyranose (PGG), a yeast derived beta-glucan, though it is facing difficulties surrounding infusion reactions in 20% of CRC patients, increased toxicity, and diminished effects when utilized with anti-inflammatory medications such as steroids (Thomas et al., 2017; Segal et al., 2016; Halstenson et al.,
2016; Bose et al. 2019). Because of these negative effects, an alternative immunomodulator, such as AHCC, could be beneficial in treating metastatic MSI-
L CRC. In this study, the effects of AHCC on CRC will be examined, as well as the underlying mechanism. It is our hope that as we further understand AHCC, we will be able to focus future studies on how this therapy can be combined with approved immunotherapies, such as PD-1 inhibitors, to improve treatment of advanced CRC.
Figure 4: Clinical responses to Pembrolizumab treatment in colorectal cancer (Le at al., 2015).
13 Our laboratory has demonstrated the effects of AHCC in the CT26 cell line, a murine-derived colon cancer commonly used to study immune mediated responses to cancer treatment. We first found that AHCC had no effect on cell growth in vitro (Figure 5) in multiple CRC cell lines. We then tested AHCC’s effects in vivo and found that it decreased tumor growth by 60% (p<0.05) (Figure 6), but had no effect in NODscid gamma (NSG), or nude mice (Figure 7). Interestingly, the CT26 cell line is thought to represent MSI-L CRC, and therefore is not expected to respond to immunotherapy (Efremova et al., 2018). Based on this initial data, we hypothesized that AHCC requires the immune system to produce its anticancer effects. Thus, this study aims to further characterize the anticancer effects of
AHCC on CRC and the underlying mechanism.
1.6
1.4
1.2 S.E.M.) - 1.0
0.8
0.6 Cal AHCC A
CT26 AHCC A Relative Growth (+/ 0.4
DLD AHCC A 0.2 HCT AHCC A 0.0 0.001 0.01 0.1 1 Drug Concentration (uM)
Figure 5: AHCC treatment in vitro. CRC cell lines at various AHCC concentrations. Standard error bars.
14 Figure 6: AHCC effects in vivo. CT26 implanted on day 1 into Balb/C mice. AHCC treatment started on day 10 at different doses (mg). (n=5 for each group) Standard error bars.
NSG 3000
2500
2000
1500 Untreated Treated Tumor Volume Tumor 1000
500
0 10 14 17 21 24 Day
Figure 7: AHCC treatment in NSG mice. CT26 implanted on day 1 into NSG mice. AHCC treatment (40mg/day) started on day 10. (n=10 for each group) Standard error bars.
15
METHODS
Mice: Balb/c mice are used in this study, as CT26 is a cell derived from this model.
Only female Balb/c mice are used in these studies, as male mice are rarely used in immune response studies, possibly due to the effects associated with their aggression and consequent stress.
AHCC Preparation: To prepare 120mg/200µL of AHCC for each mouse, 10g (1 packet) of AHCC was dissolved in 16.7mL of sterile water, and serially diluted as needed for other doses. From there, 200µL of the AHCC solution was given to each mouse by oral gavage.
Aim 1- Dosing Study: In our previous dosing experiment, 40mg of AHCC proved to be the most optimal of the doses tested (Figure 6), which is roughly equivalent to the 3g per day used in previous clinical studies (Smith et al., 2019). Because
40mg of AHCC was the highest dose tested in Balb/c mice, we wanted to determine if a dose higher than 40mg enhanced the anti-tumor effects. To determine what dose of AHCC produces the best anticancer effects, female Balb/c mice were injected with CT26 subcutaneously. Ten days after injection, the mice were treated with AHCC by oral gavage daily until the mouse no longer met ARC’s standards (ie: visible surface ulceration or tumor size impeded livelihood). The four arms were: 1) control (n=5), 2) 40mg AHCC (n=5), 3) 80mg AHCC (n=5), and 4)
16 120mg AHCC (n=5). This study determined the optimal dose of AHCC to be used in the subsequent studies. Statistical significance was calculated using a 2-tailed distribution T-Test, with p<0.05 indicating significance.
Aim 2- Long Term Study: To determine whether AHCC temporarily or permanently activates the immune system, we designed an experiment in which treatment was either given constantly, stopped and restarted, or permanently stopped. If the permanently stopped group saw an increase in tumor growth after treatment ceased, this would imply that AHCC’s effects are temporary. If this is the case, we then wanted to determine if the anti-tumor effects could be reinitiated by reintroducing AHCC in the stop/restart arm. Conversely, if AHCC’s anti-tumor effects persisted after treatment ceased, this would imply that AHCC had more permanent or long-term effects on the immune system. To determine if AHCC has long-term effects, female Babl/c mice were injected with CT26 subcutaneously and treatments began 10 days after injection. The four arms were: 1) control arm (n=9),
2) 120mg of AHCC, the optimal dose of AHCC obtained in the dosing study (n=9),
3) 120mg of AHCC until day 14, at which time treatment was temporarily stopped until day 21, when treatment was restarted (n=8), and 4) 120mg of AHCC until day
14, when treatment was permanently stopped (n=8). Each arm consisted of 10 mice initially, but if a mouse did not present a measurable tumor by day 10, the start of treatment, it was removed from the study. Mice remained in the study until their tumor became ulcerated on the surface or exceeded a volume of 3,000mm3.
17 Statistical significance was calculated using a 2-tailed distribution T-Test, with p<0.05 indicating significance.
Aim 3- Mechanism: To determine the mechanism behind AHCC’s antitumor effects, FACS analysis was performed in our previous dosing study on the tumors post-mortem. We found no difference in the number of T cells, B cells, or dendritic cells between the untreated and treated groups after several weeks of treatment
(Figure 8), which led us to hypothesize that AHCC’s effects may be acute, or more immediate, and therefore we were missing the effects on the immune system by analyzing the tumors after several weeks of treatment. To analyze AHCC’s acute mechanism of action, FACS analysis was performed on Balb/c mice treated with a single dose of 120mg of AHCC. Peripheral blood was collected either at 24 or 72 hours after treatment and FACS analysis was performed, specifically looking to identify the following: MHCII+CD11b+ (a subset of dendritic cells and macrophages), CD11b+ (monocytes), MHCII+CD11c+ (a subset of dendritic cells and macrophages), and MHCII+ (antigen presenting cells). The FACS analysis followed standard protocol, which includes washing the cells in FACS buffer and adjusting the cell number to a concentration of 500,000-1,000,000 cells/µL of sample, blocking for 20 minutes, adding 1µL of the respective antibody and incubating for 1 hour on ice in the dark, washing 3 times in FACS buffer, and finally the analysis. Each arm contained 5 mice. Statistical significance was calculated using a 2-tailed distribution T-Test, with p<0.05 indicating significance.
18 T cells B cells 50 CD3 in tumor weight 150 40 CD4 in tumor weight CD8 30 100 CD19+B220+ mg mg CD19-B220+ 20 50 10
0 0
Untreated Untreated 5 mg AHCC 5 mg AHCC 20 mg AHCC 40 mg AHCC 20 mg AHCC 40 mg AHCC
Other
100
mg 50 CD11b+MHCII- CD11b-MHCII+ CD11b+MHCII+ 0
Untreated 5 mg AHCC 20 mg AHCC 40 mg AHCC
Figure 8: FACs Analysis of Tumors. Balb/c mice were treated either not treated or treated in 5mg, 20mg, or 40mg of AHCC daily as a part of a previous dosing study. After several weeks of treatment, the mice were euthanized and their tumors analyzed for various immune cells including T cells (CD3, CD4, and CD8), B cells (CD19+B220+ and CD19-B220+), and dendritic cells (CD11B+MHCII-, CD11b-MHCII+, and CD11B+MHCII+). There was no significant difference in the various immune cell populations between treatment groups. Standard error bars.
19 RESULTS:
Aim 1- Dosing Study: To determine the optimal dose of AHCC, Balb/c mice were treated daily via oral gavage with either 0mg, 40mg, 80mg, or 120mg of AHCC
10 days after a subcutaneous injection of CT26. The 120mg dose decreased the mouse tumor volume by 58.3% (p<0.05) at day 28 compared to the control and outperformed the 40mg and 80mg, decreasing tumor volume by 19.41% and
40.02% respectively, though this was not statistically significant (p>0.05). (Figure
9). 6000
5000 Control AHCC 40 MG 4000 AHCC 80MG AHCC 120MG 3000
2000 Tumor Volume Tumor 58.3% 1000 *p<0.05
0 Day 10 Day 14 Day 17 Day 21 Day 24 Day 28 Day
Figure 9: AHCC Dosing Study. CT26 implanted on day 1 into Balb/C mice. AHCC treatment started on day 10 at different doses (n=5 for each group). Standard error bars.
Aim 2- Long-Term Study: To determine the long-term effects of AHCC on tumor growth, Balb/c mice were injected with CT26 and treated with 120mg of AHCC on day 10 post-injection. The groups were: a control arm receiving no AHCC treatment (n=9), a constant treatment group receiving AHCC from day 10 until the mice had to be euthanized due to visible surface ulceration or a tumor
20 volume exceeding 3,000mm3 (n=9), a stop/restart arm that received treatment until day 14, temporarily stopped treatment until day 21, upon which AHCC treatment resumed (n=8), and an arm that stopped treatment at day 14 permanently (n=8). On day 24, 5 mice from the control arm, 2 from the constant treatment arm, 1 from the stop/restart arm, and 3 from the permanent stop arm had to be euthanized. On day 31, 2 mice from the control arm, 4 mice from the constant treatment arm, 4 mice from the stop/restart arm, and 2 mice from the permanent stop arm had to be euthanized. To normalize the data, once a mouse was euthanized, their tumor volume on the date of euthanasia was used on subsequent days’ calculations. All mice were euthanized by day 31. While the constant treatment group was not significantly different than the control (p>0.05), the stop/restart treatment group showed 24-49% significantly decreased tumor growth than the control group on days 17-24 (p<0.05). Additionally, the permanent stop group significantly decreased tumor growth by 47% compared to the control arm on day 21 (p<0.05) (Figure 10). Ultimately, on day 31, the last day of measurement, the constant treatment and stop/restart arms increased tumor growth by 3.74% and 6.28% respectively and the permanent stop arm decreased tumor growth by 19.24%, though none of these changes were statistically significant (p>0.05). Additionally, on day 31, the stop/restart arm increased tumor growth by 2.45% and the permanent stop arm decreased tumor growth by 22.24% compared to the constant treatment arm, but these differences were also not significant (p>0.05). Finally, on day 31, the permanent stop group decreased tumor volume by 24.1% compared to the stop/restart arm, though this
21 was not statistically significant (p>0.05). The percent change in tumor size compared to the initial tumor size at day 10 was also calculated with the same normalization technique used in the volume calculations for animals euthanized, though there was no significant difference between any of the groups (p>0.05)
(Figure 11).
4000
3500
3000
2500
2000
1500 TUMOR VOLUME TUMOR 28.9% 1000 *p<0.05 500 47.0-48.8% 43.0% *p=0.03 0 *p=0.02 DAY 10 DAY 14 DAY 17 DAY 21 DAY 24 DAY 28 DAY 31 DAY
NO TREATMENT FULL TREATMENT STOP/RESTART STOP
Figure 10: AHCC Long Term Study, Change in Volume. CT26 implanted on day 1 into Balb/C mice. AHCC treatment started on day 10 at 120mg. No treatment group (n=9), constant treatment group (n=9), stop/restart group (n=8), permanently stop at day 14 group (n=8). Standard error bars.
22 7000
6000
5000
4000
3000
2000
1000 % CHANGE FROM INITIAL TUMOR VOLUME 0 DAY 10 DAY 14 DAY 17 DAY 21 DAY 24 DAY 28 DAY 31 DAY
NO TREATMENT FULL TREATMENT STOP/RESTART STOP
Figure 11: AHCC Long-Term Study, Percent Change from Initial Tumor Size at Day 10. CT26 implanted on day 1 into Balb/c mice. AHCC treatment started on day 10 at 120mg. No treatment group (n=9), constant treatment group (n=9), stop/restart group (n=8), permanently stop at day 14 group (n=8). No statistical significance. Standard error bars.
23 Survival proportions 100 Untreated 80 AHCC continuous AHCC Stop/restart 60 AHCC Stop
40
Percent survival 20
0 0 10 20 30 Time
Figure 12: AHCC Long-Term Study Survival Curve. CT26 implanted on day 1 into Balb/c mice. AHCC treatment started on day 10 at 120mg. No treatment group (n=9), constant treatment group (n=9), stop/restart group (n=8), permanently stop at day 14 group (n=8).
Aim 3- Mechanism: Comparing FACS analysis of immune cell populations in
Balb/c peripheral blood 24 hours and 72 hours post-AHCC treatment resulted in significantly lower levels of MHCII+CD11B+ and MHCII+CD11C+ cells (a subset of dendritic cells/macrophages) in the treated arm at 24 hours, but by 72 hours these levels returned to normal. CD11B+ (monocytes) and MHCII+ (antigen presenting cells) cell populations did not differ between the treated and untreated groups (Figure 13).
24 72 hr post treatment 30000 Untreated Treated 20000
10000
500 300400 cell numberscell in 100000 200 1000
CD11B+ MHCII+
MHCII+CD11b+ MHCII+CD11c+
Figure 13. FACS Analysis on Peripheral Blood. Balb/c mice were either not treated or treated with 120mg of AHCC on day 0. Their peripheral blood was collected at 24 and 72 hours and analyzed for various immune cell populations including MHCII+CD11b+ (subset of dendritic cells/macrophages), CD11B+ (monocytes), MHCII+CD11C+ (subset of dendritic cells/macrophages), and MHCII+ (antigen presenting cells). Standard error bars.
DISCUSSION:
We conducted a series of experiments to determine AHCC’s effects on
CRC. We examined AHCC in part because it also contains alpha glucans which are unique compared to other mushroom extracts. We conducted our experiments using CT26, a murine derived colon cancer cell line, which is thought to represent MSI-L CRC (Efremova et al., 2018), which has a low response rate (<5%) to approved PD-1 inhibitors, yet accounts for the majority of advanced CRC (Battaglin et al., 2018). With AHCC, we were able to induce an immune response in the MSI-L CRC model, decreasing tumor volume by 58.3%
(p<0.05) with an optimal dose of 120mg (Figure 9). This is three times the human equivalent prescribed to patients in clinical studies (Smith et al., 2019). While this was the highest dosage tested, we could not increase the AHCC concentration because past 120mg the AHCC would not dissolve in water. It is also worth
25 noting that while the 120mg arm performed the best, decreasing tumor growth by
58.3%, this is similar to the tumor reduction seen in the original dosing study in the 40mg arm which decreased tumor growth by 59.8% (Figure 6), thus 120mg may not provide significantly better benefits than 40mg, as the two doses performed similarly. As for the long-term study, the stop-restart group proved to be significantly better at slowing tumor growth than the control on days 17-24.
The stop-restart group also significantly outperformed the constant treatment group, indicating that daily dosing may not be the most effective treatment schedule (Figure 10). This is particularly novel, as previous studies on common mushroom strains such as PSK and AHCC use a daily dosing schedule, which our study indicates may not be the most effective treatment schedule. Thus, we are planning an additional dosing study to compare tumor volume and survival when AHCC is given daily, 3 times per week, twice per week, and once a week to determine an optimal dosing schedule to enhance the effects of mushroom extracts.
We hypothesize that constant AHCC treatment may be leading to tolerance or immune cell exhaustion, as our previous study’s FACS data indicates that AHCC treatment shows no difference in T cells, B cells, or dendritic cells after weeks of treatment (Figure 8). Mechanistically, the short-term effects can be seen in the FACS analysis (Figure 13) of MHCII+CD11B+ and
MHCII+CD11C+ cells, which are thought to represent a subset of dendritic cells and macrophages. These two cell populations are significantly lower in the treated arm than the untreated arm at 24 hours, but return to baseline by 72
26 hours. This could be interpreted as AHCC sending these cell populations from the periphery to tissue (ie: the tumor), and/or AHCC is causing a shift in the phenotype of dendritic cells and macrophages, perhaps to a phenotype that is associated with greater anticancer effects. This could also support our tolerance theory, as the effects on the immune system at 24 hours are not permanent and the effects on immune cell populations disappear by 72 hours. To test this theory of tolerance, an additional acute mechanistic study is underway, where the effects of various dosing schedules on the immune system will be examined at
24, 48, and 72 hours. To determine whether AHCC sends the immune cells to a tumor, an additional acute study will be performed to compare immune cell populations in peripheral blood in mice with and without tumors as well as within the tumors of tumor-bearing mice.
While these studies have provided significant insights into AHCC’s effects on CRC, numerous future studies are needed to enhance our understanding of the mechanism of action for this compound and to optimize its use in cancer patients. As previously stated, an additional dosing study is soon to begin to determine the optimal dosing schedule of AHCC. Additionally, a repeat of the short-term mechanistic study is underway to look at the effects of different dosing schedules on the immune system acutely and then later the effects AHCC has acutely when a tumor is present. The long-term mechanistic effects are currently being studied to determine what immune cells are present after sustained AHCC treatment by immunohistochemical analysis on the tumors and spleens of the euthanized mice at the end of each study. Cell markers being tested include CD3
27 (T cells), CD8 (cytotoxic T cells), F4/80 (macrophages), CD49B (natural killer cells), and SIRP Alpha (activated macrophages). We anticipate an increase in macrophages and natural killer cells, as seen in previous mushroom studies
(Ayeka et al., 2018; Lemieszek et al., 2019; Wang et al., 2015). Other planned studies include a study to identify if AHCC has any preventative effects, determining AHCC’s effects on the microbiome, and examining AHCC’s effects in
TLR2 and TLR4 knockout mice, as these receptors are thought to be one possible mechanism AHCC operates through (Daddaoua et al., 2013; Mallet et al., 2016).
Perhaps the most interesting and important future study involves combining a PD-1 inhibitor and AHCC in MSI-H (MC38) and MSI-L (CT26) cancer models (Efremova et al., 2018). Because PD-1 inhibitors have limited success in metastatic MSI-L CRC, which account for the majority of advanced
CRCs (Battaglin et al., 2018), an alternative immunotherapeutic approach is needed to combat these cancers, as 90% of advanced CRC patients, or 130,000 new patients annually, are not currently eligible for immunotherapies (Battaglin et al., 2018). Additionally, while MSI-H cancers do respond to PD-1 inhibitors at a rate of roughly 50% (Overman et al, 2017; Le et al., 2017), hopefully combining a
PD-1 inhibitor with AHCC could increase this success rate even further. We also intend to explore this combinatory approach in liver cancer cell lines, where PD-1 response rates remain low (Xu et al., 2018).
28 CONCLUSION:
In this study we found that the optimal dose of AHCC is 120mg, though a dosing schedule experiment will need to be completed to determine whether a daily, biweekly, or weekly dose produces the best results. This was deemed necessary after the long-term study showed that the stop-restart arm performed better than the constant treatment group, thus indicating that daily dosing could lead to increased tolerance or immune cell exhaustion. Mechanistically, AHCC may be initially recruiting macrophages and dendritic cells from the periphery and then diverting them elsewhere to have an effect and/or changing the phenotype of a subset population of macrophages and dendritic cells. While this study showed us the benefits of AHCC and insights into its mechanism, additional studies must be performed to determine an optimal dosing schedule, a further understanding into its short and long-term mechanisms, and the effects it can have when combined with approved PD-1 inhibitors.
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