Towards more reliable in vitro drug testing for head and neck squamous cell carcinoma via a human tumor–derived matrix

Head and neck cancer (HNSCC) is the 8th most common cancer globally. Despite improved therapy, the 5-year survival rate has remained stagnant, at approximately 50%. Current preclinical in vitro anticancer compound testing features a low predictive value. Traditionally, in vitro cancer cell studies are conducted on a plastic surface or using animal–derived extracellular matrices, thereby overlooking the important interaction between cancer cells and the human tumor microenvironment. To overcome this problem, we have developed a human tumor leiomyoma–derived matrix Myogel. The aim of this PhD project was to develop reliable in vitro methods for anticancer drug testing by providing a human TME for HNSCC cells. The first approach was to investigate whether the use of a Myogel matrix could improve the reliability of drug screening compared to conventional plastic or the typically applied mouse sarcoma–derived matrix, Matrigel. We applied a high-throughput drug screening (HTS) to test 19 anticancer compounds, targeting EGFR, MEK, or PI3K/mTOR on 12 HNSCC cell lines under five different culturing conditions; cells on a plastic surface and on top of or embedded in Myogel or Matrigel. We found that cells cultured in Myogel were less sensitive to the EGFR and MEK inhibitors than cultured in Matrigel or on plastic. Additionally, Myogel cultured cells reflected better the response rates of EGFR inhibitor clinical trials compared to the other cultures. Therefore, we concluded that Myogel improves the predictability of in vitro anticancer drug testing compared to the plastic and Matrigel. Next, we performed a high-throughput drug screen to identify synergistic combinations using a compound library (n=396) and irradiation using HNSCC cell lines cultured on Myogel. The Bcl-2/Bcl-xL inhibitor navitoclax, which emerged as the most promising radiosensitizer, exhibited a synergy with irradiation in 13 HNSCC cell lines. Additionally, the navitoclax–irradiation combination halted the cell cycle and increased the irradiation-induced apoptosis. Overall, we demonstrated that the navitoclax–irradiation combination resulted in strong synergistic antitumor effects in HNSCC cell lines, and thereby it might possess therapeutic potential for HNSCC patients. The third, we manufactured a fully human in vitro microfluidic chip assay to test immunotherapeutic agents for personalized medicine purposes for HNSCC patients. First, the assay was run using the oral tongue cancer cell line (HSC-3) embedded in a Myogel–fibrin mixture and immune cells, isolated from healthy blood samples. After successful testing, the chip was applied for freshly isolated patent cells. Immune checkpoint inhibitors, IDO1 inhibitor (NLG-919) and a PD-L1 antibody were applied to chips and the immune cell migration and cancer cell proliferation were measured over three days. We found that the immune cell migration was associated with cancer cell density. The IDO1 inhibitor increased the immune cell migration towards cancer cells in HSC-3 and in two patient samples. In the future, this assay could possibly be utilized to predict an individual patient’s immunotherapy response against HNSCC.Pään ja kaulan alueen syöpä on maailmanlaajuisesti kahdeksanneksi yleisin syöpätyyppi. Kehittyneistä hoidoista huolimatta, 5 vuoden eloonjäämisennuste on edelleen vain 50 %. Nykyisten prekliinisten syöpälääkekokeiden ennustearvo on alhainen. Solututkimukset tehdään tavallisesti muovialustalla tai käyttämällä kaupallisia eläinperäisiä väliaineita, kuten hiiriperäistä Matrigeeliä. Nämä menetelmät jäljittelevät kuitenkin heikosti ihmisyöpäsolujen kasvuympäristöä. Parantaaksemme solukokeiden ennustettavuutta, olemme kehittäneet ihmisen kohdun myoomasta valmistetun väliaineen Myogeelin. Tämän väitöskirjatyön tavoitteena oli kehittää luotettavampia in vitro -menetelmiä tarjoamalla syöpäsoluille ominaisempi kasvuympäristö. Ensimmäiseksi tutkimme, voisiko Myogeelin käyttö parantaa lääkeseulonnan luotettavuutta muovialustaan tai Matrigeeliin verrattuna. Tutkimuksessa testattiin 19 täsmälääkkeen (EGFR:n, MEK:n ja PI3K / mTOR:n estäjien) tehoa kahdellatoista pään ja kaulan alueen syöpäsolulinjalla viidessä eri kasvuympäristössä; muovin päällä, Myogeelillä ja Matrigeelillä päällystetyillä levyillä ja geelien sisällä. Myogeelin päällä ja sisällä kasvatetut syöpäsolut reagoivat huomattavasti heikommin EGFR:n ja MEK:n estäjiin kuin Matrigeelissä tai muovin päällä kasvatetut solut. Lisäksi Myogeelissä tehdyt kokeet vastasivat muita koeolosuhteita paremmin kliinisiä EGFR:n estäjillä tehtyjä lääkekokeita. Tutkimustuloksien perusteella Myogel parantaa in vitro ‑syöpälääketestausten kliinistä ennustettavuutta. Seuraavaksi tutkimme suuritehoisella seulonnalla uusia säde- ja lääkehoitoyhdistelmiä. Tutkimuksessa käytettiin lääkekirjastoa (n = 396) ja sädehoitoa Myogeelissä viljellyille pään ja kaulan alueen syöpäsoluille. Tutkimuksessa osoitettiin lupaavimman lääkekandidaatin, Bcl-2/Bcl-xL estäjän, navitoklaksin ja sädehoidon yhdistelmällä olevan synerginen vaikutus kolmessatoista testatussa solulinjassa. Lisäksi yhdistelmä pysäytti syöpäsolujen jakautumisen ja aiheutti apoptoosia. Tutkimuksen perusteella navitoklaksi-sädehoitoyhdistelmällä saattaisi olla potentiaalia pään ja kaulan alueen syövän hoidossa. Kolmantena aiheena oli kehittää ihmisperäinen mikrofluidinen sirumääritys yksilöidyn immunoterapiavasteen testaukseen. Ensiksi siru testattiin käyttäen kielisyöpäsolulinjaa (HSC-3) ja terveiden verenluovuttajien immuunisoluja. Syöpäsolut kasvatettiin sirussa Myogeeli-fibriini-väliaineessa. Immuunijärjestelmän tarkastuspiste-estäjiä, IDO1-estäjää (NLG-919) ja PD-L1-vasta-ainetta lisättiin siruille, minkä jälkeen immuunisolujen liikkumista sekä syöpäsolujen jakautumista mitattiin kolmen päivän ajan. Seuraavaksi sirumäärityksen suoritettiin potilaista eristetyille syöpäsoluille ja veren immuunisoluille. Tutkimuksemme osoittivat syöpäsolujen määrän vaikuttavan immuunisolujen liikkumiseen. IDO1-estäjä lisäsi immuunisolujen liikkumista syöpäsoluja kohden kahdella potilasnäytteellä sekä HSC-3-soluilla. Määritystä voitaisiin mahdollisesti tulevaisuudessa käyttää apuna potilaan immunoterapiavasteen ennustamisessa

Effect of Sex Steroid Hormones on Tongue Cancer Cells In Vitro

Background: Tongue cancer is more common in men than in women. Yet the effects of sex steroid hormones on the behaviour of oral tongue squamous cell carcinoma (OTSCC) are not well known. Matrix metalloproteinase 8 (MMP8) is expressed in OTSCC and can degrade estrogen receptors (ERs). Materials and Methods: Western blot was used to examine the levels of ER beta in OTSCC cell lines (HSC-3 and SCC-25). We evaluated the effects of estradiol and dihydrotestosterone (DHT) on HSC-3 and SCC-25 cell migration, invasion and viability. The effect of estradiol on the invasion of MMP8-overexpressing (MMP8(+)) and empty vector HSC-3 cells was examined using 3D spheroid invasion assay. Results: Both HSC-3 and SCC-25 cells expressed ER beta. In scratch assay, estradiol, but not DHT, reduced the migration and invasion of HSC-3 and SCC-25 cells. MMP8(+) HSC-3 cells showed weaker invasion than empty vector cells, in line with previous reports. However, MMP8 overexpression did not alter the effect of estradiol on HSC-3 cell invasion in spheroid assay. Conclusion: Estradiol inhibited the migration and invasion of OTSCC cells, whereas DHT had no effect. Our data suggest that MMP8 does not modulate the effect of estradiol in OTSCC cells. However, the sex difference in OTSCC incidence might partly be due to protective actions of estradiol in epithelial cell carcinogenesis.Peer reviewe

Organotypic three-dimensional assays based on human leiomyoma-derived matrices

Alongside cancer cells, tumours exhibit a complex stroma containing a repertoire of cells, matrix molecules and soluble factors that actively crosstalk between each other. Recognition of this multifaceted concept of the tumour microenvironment (TME) calls for authentic TME mimetics to study cancer in vitro. Traditionally, tumourigenesis has been investigated in non-human, three-dimensional rat type I collagen containing organotypic discs or by means of mouse sarcoma-derived gel, such as Matrigel (R). However, the molecular compositions of these simplified assays do not properly simulate human TME. Here, we review the main properties and benefits of using human leiomyoma discs and their matrix Myogel for in vitro assays. Myoma discs are practical for investigating the invasion of cancer cells, as are cocultures of cancer and stromal cells in a stiff, hypoxic TME mimetic. Myoma discs contain soluble factors and matrix molecules commonly present in neoplastic stroma. In Transwell, IncuCyte, spheroid and sandwich assays, cancer cells move faster and form larger colonies in Myogel than in Matrigel (R). Additionally, Myogel can replace Matrigel (R) in hanging-drop and tube-formation assays. Myogel also suits three-dimensional drug testing and extracellular vesicle interactions. To conclude, we describe the application of our myoma-derived matrices in 3D in vitro cancer assays. This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.Peer reviewe

Adipose-Derived Mesenchymal Stem Cells do not Affect the Invasion and Migration Potential of Oral Squamous Carcinoma Cells

Mesenchymal stem cells (MSCs) are commonly isolated from bone marrow and adipose tissue. Depending on the tissue of origin, MSCs have different characteristics and physiological effects. In various cancer studies, MSCs have been found to have either tumor-promoting or tumor-inhibiting action. This study investigated the effect of adipose tissue-MSCs (AT-MSCs) and bone marrow-MSCs (BM-MSCs) on global long interspersed nuclear element-1 (LINE-1) methylation, the expression level of microenvironment remodeling genes and cell proliferation, migration and invasion of oral tongue squamous cell carcinoma (OTSCC). Additionally, we studied the effect of human tongue squamous carcinoma (HSC-3)-conditioned media on LINE-1 methylation and the expression of microenvironment remodeling genes in AT-MSCs and BM-MSCs. Conditioned media from HSC-3 or MSCs did not affect LINE-1 methylation level in either cancer cells or MSCs, respectively. In HSC-3 cells, no effect of MSCs-conditioned media was detected on the expression of ICAM1, ITGA3 or MMP1. On the other hand, HSC-3-conditioned media upregulated ICAM1 and MMP1 expression in both types of MSCs. Co-cultures of AT-MSCs with HSC-3 did not induce proliferation, migration or invasion of the cancer cells. In conclusion, AT-MSCs, unlike BM-MSCs, seem not to participate in oral cancer progression

Adipose-Derived Mesenchymal Stem Cells do not Affect the Invasion and Migration Potential of Oral Squamous Carcinoma Cells

Mesenchymal stem cells (MSCs) are commonly isolated from bone marrow and adipose tissue. Depending on the tissue of origin, MSCs have different characteristics and physiological effects. In various cancer studies, MSCs have been found to have either tumor-promoting or tumor-inhibiting action. This study investigated the effect of adipose tissue-MSCs (AT-MSCs) and bone marrow-MSCs (BM-MSCs) on global long interspersed nuclear element-1 (LINE-1) methylation, the expression level of microenvironment remodeling genes and cell proliferation, migration and invasion of oral tongue squamous cell carcinoma (OTSCC). Additionally, we studied the effect of human tongue squamous carcinoma (HSC-3)-conditioned media on LINE-1 methylation and the expression of microenvironment remodeling genes in AT-MSCs and BM-MSCs. Conditioned media from HSC-3 or MSCs did not affect LINE-1 methylation level in either cancer cells or MSCs, respectively. In HSC-3 cells, no effect of MSCs-conditioned media was detected on the expression of ICAM1, ITGA3 or MMP1. On the other hand, HSC-3-conditioned media upregulated ICAM1 and MMP1 expression in both types of MSCs. Co-cultures of AT-MSCs with HSC-3 did not induce proliferation, migration or invasion of the cancer cells. In conclusion, AT-MSCs, unlike BM-MSCs, seem not to participate in oral cancer progression

The critical effects of matrices on cultured carcinoma cells : Human tumor-derived matrix promotes cell invasive properties

The interaction between squamous cell carcinoma (SCC) cells and the tumor microenvironment (TME) plays a major role in cancer progression. Therefore, understanding the TME is essential for the development of cancer therapies. We used four (primary and metastatic) head and neck (HN) SCC cell lines and cultured them on top of or within 5 matrices (mouse sarcoma-derived Matrigel (R), rat collagen, human leiomyoma-derived Myogel, human fibronectin and human fibrin). We performed several assays to study the effects of these matrices on the HNSCC behavior, such as proliferation, migration, and invasion, as well as cell morphology, and molecular gene profile. Carcinoma cells exhibited different growth patterns depending on the matrix. While fibrin enhanced the proliferation of all the cell lines, collagen did not. The effects of the matrices on cancer cell migration were cell line dependent. Carcinoma cells in Myogel-collagen invaded faster in scratch wound invasion assay. On the other hand, in the spheroid invasion assay, three out of four cell lines invaded faster in Myogel-fibrin. These matrices significantly affected hundreds of genes and a number of pathways, but the effects were cell line dependent. The matrix type played a major role in HNSCC cell phenotype. The effects of the ECMs were either constant, or cell line dependent. Based on these results, we suggest to select the most suitable matrix, which provides the closest condition to the in vivo TME, in order to get reliable results in in vitro experiments.Peer reviewe

In vitro humanized 3D microfluidic chip for testing personalized immunotherapeutics for head and neck cancer patients

Objectives: Immunotherapy and personalized medicine therapeutics are emerging as promising approaches in the management of head and neck squamous cell carcinoma (HNSCC). In spite of that, there is yet no assay that could predict individual response to immunotherapy. Methods: We manufactured an in vitro 3D microfluidic chip to test the efficacy of immunotherapy. The assay was first tested using a tongue cancer cell line (HSC-3) embedded in a human tumour-derived matrix "Myogel/fibrin" and immune cells from three healthy donors. Next, the chips were used with freshly isolated cancer cells, patients' serum and immune cells. Chips were loaded with different immune checkpoint inhibitors, PD-L1 antibody and IDO 1 inhibitor. Migration of immune cells towards cancer cells and the cancer cell proliferation rate were evaluated. Results: Immune cell migration towards HSC-3 cells was cancer cell density dependent. IDO 1 inhibitor induced immune cells to migrate towards cancer cells both in HSC-3 and in two HNSCC patient samples. Efficacy of PD-L1 antibody and IDO 1 inhibitor was patient dependent. Conclusion: We introduced the first humanized in vitro microfluidic chip assay to test immunotherapeutic drugs against HNSCC patient samples. This assay could be used to predict the efficacy of immunotherapeutic drugs for individual patients.Peer reviewe

High-throughput compound screening identifies navitoclax combined with irradiation as a candidate therapy for HPV-negative head and neck squamous cell carcinoma

Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and the development of resistance. Therefore, new molecular-targeted therapies are urgently needed to be integrated into existing treatment regimens. Here, we performed a high-throughput compound screen to identify a synergistic interaction between ionizing radiation and 396 anticancer compounds. The assay was run using five human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) cell lines cultured on the human tumor-derived matrix Myogel. Our screen identified several compounds with strong synergistic and antagonistic effects, which we further investigated using multiple irradiation doses. Navitoclax, which emerged as the most promising radiosensitizer, exhibited synergy with irradiation regardless of the p53 mutation status in all 13 HNSCC cell lines. We performed a live cell apoptosis assay for two representative HNSCC cell lines to examine the effects of navitoclax and irradiation. As a single agent, navitoclax reduced proliferation and induced apoptosis in a dose-dependent manner, whereas the navitoclax-irradiation combination arrested cell cycle progression and resulted in substantially elevated apoptosis. Overall, we demonstrated that combining navitoclax with irradiation resulted in synergistic in vitro antitumor effects in HNSCC cell lines, possibly indicating the therapeutic potential for HNSCC patients.Peer reviewe

Human Tumor–Derived Matrix Improves the Predictability of Head and Neck Cancer Drug Testing

In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–derived matrix “Myogel” to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo–relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma–derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. However, we found a similar response to the PI3K/mTOR inhibitors under all culturing conditions. Cells grown on Myogel more closely resembled the response rates reported in EGFR-inhibitor monotherapy clinical trials. Our findings suggest that a human tumor matrix improves the predictability of in vitro anticancer drug testing compared to current 2D and MSDM methods

Human Tumor–Derived Matrix Improves the Predictability of Head and Neck Cancer Drug Testing

In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–derived matrix “Myogel” to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo–relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma–derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. However, we found a similar response to the PI3K/mTOR inhibitors under all culturing conditions. Cells grown on Myogel more closely resembled the response rates reported in EGFR-inhibitor monotherapy clinical trials. Our findings suggest that a human tumor matrix improves the predictability of in vitro anticancer drug testing compared to current 2D and MSDM methods