Caloric restriction mimetics (CRMs), compounds that mimic the biochemical effects of nutrient deprivation, administered via systemic route promote antitumor effects through the induction of autophagy and the modulation of the immune microenvironment; however, collateral effects due to metabolic changes and the possible weight loss might potentially limit their administration at long term. Here, we investigated in mice local administration of CRMs via aerosol to reduce metastasis implantation in the lung, whose physiologic immunosuppressive status favors tumor growth. Hydroxycitrate, spermidine, and alpha-lipoic acid, CRMs that target different metabolic enzymes, administered by aerosol, strongly reduced implantation of intravenously injected B16 melanoma cells without overt signs of toxicity, such as weight loss and changes in lung structure. Cytofluorimetric analysis of lung immune infiltrates revealed a significant increase of alveolar macrophages and CD103+ dendritic cells in mice treated with CRMs that paralleled an increased recruitment and activation of both CD3 T lymphocytes and NK cells. These effects were associated with the upregulation of genes related to M1 phenotype, as IL-12 and STAT-1, and to the decrease of M2 genes, as IL-10 and STAT-6, in adherent fraction of lung immune infiltrate, as revealed by real-time PCR analysis. Thus, in this proof-of-principle study, we highlight the antitumor effect of CRM aerosol delivery as a new and noninvasive therapeutic approach to locally modulate immunosurveillance at the tumor site in the lung.
The study of cancer metabolism is now receiving substantial attention for its implications in the biology of cancer and the possibility to find new therapeutic interventions. Recently, caloric restriction mimetics (CRMs), compounds mimicking the biochemical effects of nutrient deprivation, have revealed antitumor properties [
The biological activity of these compounds mostly relies on their ability to induce autophagy, shaping the tumor microenvironment (TME), and promote anticancer immunosurveillance [
Since lungs are constantly exposed to inhaled antigens, these organs represent a particularly immunosuppressive milieu to limit excessive immune response. It has been speculated that cancer cells can harness this unique environment for their implantation and growth, explaining, at least in part, the high incidence of lung metastases arising from several types of tumors [
Aerosolization is an efficient and noninvasive method of delivering molecules to the lung in order to improve local tissue concentration, limiting potential adverse effects induced by a systemic administration, and we previously demonstrated its usefulness to modulate lung microenvironment [
In the present study, we evaluated local administration by aerosol delivery of hydroxycitrate, spermidine, and alpha-lipoic acid to reduce tumor implantation in the lung of mice intravenously injected with B16 melanoma cells, as a new therapeutic approach to locally modulate antitumor immune response.
B16 mouse melanoma cells (American Type Culture Collection (ATCC), Rockville, MD, USA) and N202.1A cells, derived from a spontaneous mammary carcinoma in an FVB-neuN transgenic mouse [
Cell lines were authenticated by the Fragment Analysis Facility at Fondazione IRCCS Istituto Nazionale dei Tumori (Milan, Italy) using the GenePrint 10 System (Promega, Madison, WI, USA), and cultures were regularly tested for Mycoplasma by using the MycoAlert Plus Kit (Lonza Group Ltd., Basel, Switzerland). Hydroxycitrate, spermidine, and alpha-lipoic acid were purchased from Sigma-Aldrich. 51Cr (1 mCi) was purchased from PerkinElmer (Waltham, MA, USA, NEZ030S001MC).
Female C57BL/6 and FVB mice, aged 6-8 weeks (Charles River Laboratories, Calco, Italy), were maintained in laminar flow rooms at constant temperature and humidity, with food and water given ad libitum. Mice were treated with alpha-lipoic acid (10 mg/kg), spermidine (5 mg/kg) (5 days/week at 12 h intervals), and hydroxycitrate (14 mg) (5 days/week) dissolved in 5 ml of saline starting 1 day after the intravenous (i.v.) injection of
The experimental protocols were carried out in accordance with the Italian law D.Lgs. 26/2014, and animal experimentation was performed following the guidelines drawn up by Fondazione IRCCS Istituto Nazionale dei Tumori Institutional Animal Welfare Body according to Workman et al. [
To exclude any effects of aerosolized CRM molecules on the architecture and structure of the lung parenchyma, lung samples were analyzed as described [
To analyze autophagy induced by CRM aerosolization, immunohistochemical analyses were performed to detect LC3B molecule. IHC was carried out on formalin-fixed paraffin-embedded lung sections using a protocol previously described with slightly modifications [
Isolation of lung immune cells was performed as described [
To analyze immune lung infiltration, lung suspensions were stained as previously described [
RNA was isolated using QIAzol (QIAGEN) from adherent cells (containing macrophage/myeloid-derived cells) according to the manufacturer’s instructions. Reverse transcription was performed using a High-Capacity RNA-to-cDNA Kit (Applied Biosystems-Thermo Fisher Scientific). Real-time PCR was performed using TaqMan® Fast Universal PCR Master Mix (Applied Biosystems-Thermo Fisher Scientific) and SDS 2.4 on a 7900HT Fast Real-Time PCR System (Applied Biosystems-Thermo Fisher Scientific), as we previously described [
The ability of effector immune cells from the lung immune infiltrates of mice to promote antitumor activity was evaluated by measuring cytotoxic activity of nonadherent cells obtained from the lung suspensions on 51Cr-B16 target cells as described [
Differences among groups were compared using a two-tailed unpaired Student’s
CRMs, whose antitumor effect has been demonstrated in different preclinical models [
To evaluate whether aerosolized CRMs are able to reach the alveolar space and to control metastasis implantation, female C57BL/6 mice, intravenously (i.v.) injected with murine B16 melanoma cells, were treated with hydroxycitrate, spermidine, and alpha-lipoic acid. B16 cell is a low immunogenic tumor that establishes a highly immunosuppressive microenvironment recruiting tumor-infiltrating macrophages (TAMs) and MDSC [
The combination of hydroxycitrate, spermidine, and alpha-lipoic acid was chosen to concomitantly target different metabolic enzymes, based on the complexity of the aerobic glycolytic pathway and on previous published results that demonstrated the superior efficacy of combinations of these compounds than single agents alone in preclinical cancer models [
Treatments with aerosolized CRMs were well-tolerated, as indicated by the absence of signs of toxicity, such as hunching, ruffled fur, and difficulty breathing. Moreover, no weight loss was observed in CRM-treated mice (Figure
Effects of CRM aerosolization on body weight, on the growth of experimental B16 lung metastases, and on lung histology. Body weights (a) and number of macroscopic lung metastatic foci (b) of mice (9-10 mice/group) treated starting 1 day after i.v. injection of B16 melanoma cells with aerosolized saline or combined CRMs for 3 weeks. Representative images showing histopathological evaluation of hematoxylin- and eosin-stained lung tissue sections (c) and IHC analysis of LC3B staining in tumor nodules (d) (magnification: ×200) of saline and CRM-treated mice.
Inducing autophagy, CRMs have been demonstrated to influence tumor growth by targeting cancer cell metabolism. This activity has been demonstrated to play dual effects as it not only prevents tumor initiation but also promotes tumor progression by assisting in hypoxia-induced switch to anaerobic glycolysis [
Cytofluorimetric analysis of immune infiltrate obtained after tumor-bearing lung enzymatic digestion showed a significant increase of alveolar macrophages (AMs) (FL-1+CD11c+ cells) and CD103+ dendritic cells (DCs) (CD11b-CD103+CD11c+ cells) in mice treated with CRM aerosol as compared to control group (Figure
Modification of lung immune contexture induced by CRM aerosolization. Bars (
We then evaluated whether the reduced tumor growth and the increase of APCs were associated with changes in the expression of M1/M2 genes in the lung microenvironment. Suspensions obtained from lung enzymatic digestion were seeded in culture plates to separate adherent cells, which contain macrophages and myeloid cells, from the floating counterpart, mainly constituted by effector cells. Real-time PCR analysis performed on mRNA extracted from the adherent cell fraction revealed a significant upregulation of IL-12, TNF-
The reduction of M2 polarization observed in lung immune infiltrate is in line with the recently published results that demonstrated how intermittent fasting promotes the reduction of TAM polarization and immunosuppressive activity through the inactivation of JAK1/STAT3 pathway in murine models of colon cancer [
We also analyzed the recruitment and the activation status of immune cell population that can directly kill the tumor, NK, and T cells. Flow cytometry analysis of lung immune infiltrate revealed a significant expansion of both NK cells and T lymphocytes (Figure
Effects of CRM aerosolization on the recruitment and activation of lung antitumor effector cells. Bars (
In this proof-of-concept study, we investigated the possibility to administer CRMs at the tumor site in the lung of female mice by aerosol, revealing that aerosolized CRMs can reach the bronchoalveolar space and exert antitumor activity. A possible limitation of present study is the use of only female mice, because it is known that constitutive autophagy can be different between male and female due to the biological effect of sex hormones [
In conclusion, this strategy may represent a noninvasive therapeutic approach to locally activate adaptive and innate immunosurveillance, while reducing the toxic effects related to CRM systemic administration.
The data obtained from FACS and real-time PCR analyses used to support the findings of this study are available from the corresponding author upon request.
The authors declare no potential conflicts of interest.
The authors thank Mrs. G. Abolafio of Flow Cytometry service for the technical assistance and Mrs L. Mameli for the secretarial assistance. This work was supported by Piano di Sostegno alla Ricerca 2017 Università degli Studi di Milano and by AIRC (Associazione Italiana per la Ricerca sul Cancro) (Id. 15190). F.B. is supported by a fellowship from the “Fondazione Umberto Veronesi” 2017-2018.
Supplementary Figure: modification of APC populations induced by CRM aerosolization in the N202.1A tumor.