Introduction

Cutaneous squamous cell carcinoma (cSCC), which originates from epidermal keratinocytes, is the most common metastatic skin cancer and its incidence is increasing worldwide1. The estimated metastasis rate of cSCC is 3–5%, and the prognosis of patients with metastatic disease is poor, as over 70% of patients with metastatic cSCC will die within 3 years2,3. Furthermore, cSCC has been estimated to cause 20% of skin cancer‒related mortality1. A UV-induced premalignant lesion, actinic keratosis may develop to malignant cSCC in situ (Bowen’s disease) and finally to invasive and metastatic cSCC4. The major risk factor for cSCC is long-term exposure to UV-radiation. Other risk factors for cSCC development and progression are chronic inflammation, chronic dermal ulcers, and immunosuppression4. The complement system is a part of both innate and acquired immune systems5,6. The complement system may be activated through classical, lectin, or alternative pathways and all of these pathways converge into cleavage of C3 and activation of terminal pathway and formation of lytic membrane attack complex7. C1s is a part of classical pathway initiating C1qr2s2 complex. Binding of C1q to target molecule induces autocatalytic activation of the serine proteinase C1r, which in turn activates another serine proteinase C1s8. There is also increasing evidence that components of C1 complex exert cancer-promoting functions and play a role in cancer progression independently of the complement system activation in a non-canonical manner7,9,10. C1q has been noted to be expressed in the microenvironment of several human malignant tumors and to contribute to tumor progression without activation of complement classical pathway11.

We have previously shown marked upregulation of C1s expression in tumor cells in cSCC in vivo and in cSCC cell lines in culture12. Knockdown of C1s expression resulted in decreased viability and growth of cSCC cells by promoting apoptosis both in culture and in vivo. C2 or C4 was not present in cSCC cultures indicating non-canonical function of C1s in cSCC cells13.

In this study, we have investigated the effect of C1s targeted antibodies TNT003 and TNT005 on cSCC cell growth and viability. Treatment of cSCC cells with TNT003 or TNT005 significantly inhibited their growth and promoted apoptosis. These data demonstrate that TNT003 and TNT005 are potent inhibitors of cSCC cell growth in culture and provide the rationale for additional studies in relevant in vitro and in vivo disease models to support the development of C1s targeted inhibitors for the treatment of locally advanced and metastatic cSCCs.

Materials and methods

Ethical issues

The use of tumor derived SCC cell lines was approved by the Ethics Committee of the Hospital District of Southwest Finland (187/2006). All participants gave their written informed consent, and the study was performed with the permission of Turku University Hospital, according to the Declaration of Helsinki.

C1s targeting antibodies TNT003 and TNT005

TNT003 and TNT005 were provided by Sanofi. Both antibodies inhibit the classical complement pathway. TNT003 binds to both the active and inactive forms of C1s. In contrast, TNT005 is highly specific for the active form. They are both murine IgG2a antibodies identified using the traditional hybridoma technology, as previously described14,15. Briefly, mice were immunized with human active C1s protein and a hybridoma library was generated and screened. Antibodies were purified from hybridoma supernatants using Protein A chromatography. The control antibody IgG 068 (IgG) is a mutated version of TNT003 that lacks target binding.

Cell cultures

Primary non-metastatic (UT-SCC-12A, UT-SCC-91, UT-SCC-105, UT-SCC-111 and UT-SCC-118) and metastatic (UT-SCC-7, UT-SCC-59A and UT-SCC-115) cSCC cell lines were established from surgically removed cSCCs in Turku University Hospital12,16,17 (Supplementary Table S1). cSCC cells were cultured as previously described12. The authenticity of all cSCC cell lines has been verified by short tandem repeat profiling16. The immortalized non-tumorigenic human keratinocyte–derived cell line (HaCaT) was kindly provided by Dr Norbert Fusenig (Deutsche Krebsforschungszentrum, Heidelberg, Germany). HaCaT cells were cultured as previously described13. For antibody treatment, indicated concentrations of control IgG and C1s targeting antibodies TNT003 and TNT005 were added to cSCCs cell cultures in serum free conditions for different periods of time.

Western blotting

For western blot analysis, samples were fractionated in 10% SDS–polyacrylamide gel and transferred onto nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA), as previously described12. Production of C1r and C1s by cSCC cells was determined by western blotting analysis of conditioned media or total cell lysates using specific polyclonal rabbit anti-C1s (HPA018852; Sigma-Aldrich) and anti-C1r (HPA001551; Sigma-Aldrich) antibodies. Cell lysates were also analyzed with antibodies specific for phosphorylated protein kinase B (Akt), phosphorylated extracellular signal–regulated kinase (ERK)-1/2, total ERK1/2 (9271S, 9101, and 9102, respectively; all from Cell Signaling Technology, Beverly, MA), and total Akt (sc-1618, Santa Cruz Biotechnology, Santa Cruz, CA). TIMP-1 (Ab-1; Merck Millipore) or β-actin (A1978; Sigma-Aldrich) antibody was used to determine protein loading.

Cell growth assay

Cells were seeded (7.5 × 103 cells/well) on 96-well plates. The IncuCyte S3 real-time cell imaging system (Essen BioScience, Ann Arbor, MI) was used to study the growth of cSCC cells. Images were taken every 2 h by IncuCyte S3, and the relative confluence was analyzed by the instrument. Experiments were carried out with 5–8 parallel wells at every time point with six cSCC cell lines (UT-SCC-59A, -115, -12A, -105, -118 and -7).

Cell viability assay

Cutaneous SCC (1.0 × 104 cells/well) or HaCaT (7.5 × 103 cells/well) cells were seeded on 96-well plates. The number of viable cells was determined using a Cell Counting Kit-8 (CCK-8, Do**do, Japan). The experiments were carried out with 6–8 parallel wells in every time point with six cSCC cell lines (UT-SCC-59A, -115, -12A, -105, -118 and -7) and non-tumorigenic human keratinocyte–derived HaCaT cells.

Apoptosis assays

Apoptotic cells were detected after 24 h treatment with antibodies by using In Situ Cell Death Detection Kit (Roche). Number of TUNEL positive cells was counted from 4 parallel image fields / cell line using 20 × objective and compared to total cell number visualized by Hoechst 33342 (Invitrogen). The experiments were carried out with seven cSCC cell lines (UT-SCC-59A, -115, -12A, -91, -105, -118 and -7).

Results

C1s and C1r expression by cSCC cell lines

The production of C1s and C1r by cSCC cell lines was determined by western blotting of conditioned media (Fig. 1A) and cell lysates (Fig. 1B). The secretion of C1s to cell culture media was noted in all cSCC cell lines examined (Fig. 1A). Most prominent secretion of C1s was detected in two metastatic cSCC cell lines UT-SCC-7 and -59A and in a primary non-metastatic cSCC cell line UT-SCC-111 (Fig. 1A). The levels of C1s were also detected in whole cell lysates (Fig. 1B) and in UT-SCC-115 and UT-SCC-91 the intracellular levels of C1s were higher than the secreted levels of C1s compared to other cSCC cell lines (Fig. 1A,B). C1r was secreted to the medium or noted as intracellularly in the majority of cSCC cell lines indicating the possibility to activate C1s (Fig. 1).

Figure 1
figure 1

C1s and C1r expression in cutaneous squamous cell carcinoma (cSCC) cells. A Conditioned media and B total cell lysates of non-metastatic (non-mcSCC) and metastatic (mcSCC) cSCC cell lines were collected from cell growth experiments and analyzed by western blotting. The levels of C1s and C1r are shown. TIMP-1 (A) and β-actin (B) were determined as the loading controls. Quantitations of the western blots corrected for loading controls are shown below the panels.

Full size image

C1s targeted antibodies TNT003 and TNT005 inhibit growth and viability of cSCC cells

C1s was targeted with antibodies TNT003 and TNT005 to investigate the effect of C1s in cSCC cell growth and viability. The dose response of the effect of the antibodies on cSCC cell viability was investigated (Supplementary Fig. S1) and the maximum concentration 250 µg/mL was selected based on previous publications with other cell lines18. Treatment of metastatic cSCC cell lines UT-SCC-59A and UT-SCC-115 with TNT003 and TNT005 (250 µg/mL) significantly inhibited the growth (Fig. 2A,B,D,E) and viability (Fig. 2C,F) of cSCC cells compared to control antibody (IgG). TNT003 and TNT005 (250 µg/mL) also inhibited the growth of primary UT-SCC-105, UT-SCC-12A, UT-SCC-118 and metastatic UT-SCC-7 cell lines (Supplementary Fig. S2). Additionally, TNT003 inhibited the growth of UT-SCC-115, UT-SCC-118 and UT-SCC-7 already at 200 µg/mL concentration (Supplementary Fig. S2A,D,E). Furthermore, inhibition of the growth of UT-SCC-7 cell line with TNT005 was detected already at 200 µg/mL concentration (Supplementary Fig. S2E). TNT003 and TNT005 (200µg/mL) inhibited the viability of UT-SCC-12A, UT-SCC-118, UT-SCC-105 and UT-SCC-7 cell lines (Supplementary Fig. S3).

Figure 2
figure 2

C1s targeted antibodies TNT003 and TNT005 inhibit the growth and viability of cutaneous squamous cell carcinoma (cSCC) cells. A, B Metastatic cSCC cell lines UT-SCC-59A and D, E UT-SCC-115 (7.5 × 103 cells/well) were plated on 96-well plates. Control antibody (IgG) and C1s targeting antibodies TNT003 and TNT005 were added to cells under serum free conditions. The IncuCyte S3 real-time cell imaging system was used to study the growth of cSCC cells and the relative confluence was analyzed by the instrument (n = 6–8). C UT-SCC-59A and F UT-SCC-115 cells (1.0 × 104 cells/well) were plated on 96-well plates. Control antibody (IgG) and C1s targeting antibodies TNT003 and TNT005 were added to cells in serum free conditions. The number of cSCC cells was determined at time points indicated using CCK-8 assay (n = 6–8). *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.

Full size image

The effect of TNT003 and TNT005 on the expression of C1s and C1r was investigated by western blotting of lysates of cSCC cells treated with the antibodies for 72 h (Supplementary Fig. S4). The expression of C1s and C1r was not markedly regulated by C1s targeted antibodies TNT003 or TNT005 in cSCC cells (Supplementary Fig. S4).

Immortalized non-tumorigenic keratinocyte–derived cell line (HaCaT) was utilized to determine the effect of TNT003 and TNT005 (250 µg/mL) on viability of non-malignant keratinocyte-derived cells. The production of C1s and C1r by HaCaT and UT-SCC-59A cells was examined by western blotting of conditioned media (Supplementary Fig. S5A) and cell lysates (Supplementary Fig. S5B). No expression was noted in HaCaT cells, whereas the cSCC cell line UT-SCC-59A used as positive control showed expression of both C1s and C1r (Supplementary Fig. S5A,B). Treatment with TNT003 or TNT005 had no effect on the viability of HaCaT cells (Supplementary Fig. S5C), whereas treatment of UT-SCC-59A cells in parallel with TNT003 and TNT005 (250 µg/mL) significantly inhibited their viability (Supplementary Fig. S5D).

C1s targeted antibodies TNT003 and TNT005 promote apoptosis of cSCC cells

Knockdown of C1s has been shown to promote apoptosis of cSCC cells in culture and in vivo12. Therfore, we investigated, whether TNT003 and TNT005 could induce apoptosis of cSCC cells. The representative images of these experiments with cSCC cell lines are shown in Fig. 3A and Supplementary Fig S6. Increased number of TUNEL‐positive apoptotic cSCC cells was detected after 24 h treatment with 250 µg/mL C1s targeting antibodies TNT003 and TNT005 compared to control antibody (IgG) (Fig. 3B).

Figure 3
figure 3

C1s targeted antibodies TNT003 and TNT005 promote apoptosis of cutaneous squamous cell carcinoma (cSCC) cells. A Representative images of Hoechst and TUNEL stainings for metastatic cSCC cell lines UT-SCC-59A and UT-SCC-115 are shown. Scale bar 100 µm. B Seven cSCC cell lines (UT-SCC-59A, -115, -12A, -91, -105, -118 and -7) were treated with control antibody (IgG) and C1s targeting antibodies TNT003 and TNT005 (250 µg/mL) for 24 h under serum free conditions, apoptotic cells were detected with TUNEL staining, and the relative number of TUNEL‐positive cells was counted (n = 7). *p < 0.05, ***p < 0.001, Student's t-test.

Full size image

C1s targeted antibodies TNT003 and TNT005 inhibit ERK1/2 and Akt signaling pathways in cSCC cells

Previously, C1s knockdown with specific siRNAs has been demonstrated to decrease the phosphorylation of cSCC cell growth and viability related signaling pathway molecules ERK1/2 and Akt12. Therefore, we studied the effect of TNT003 and TNT005 on these signaling pathways. Analysis of cell lysates by western blotting after 48 h treatment of UT-SCC-115 (Fig. 4A) and UT-SCC-118 (Fig. 4B) cell cultures with 250 µg/mL control antibody (IgG), TNT003 and TNT005 showed that the levels of phosphorylated Akt and phosphorylated ERK1/2 were decreased, indicating that C1s targeting antibodies inhibit the activation of the PI3K and ERK1/2 signaling pathways. Interestingly, IgG was detected in cell lysates of cSCC cells treated with control IgG, TNT003 and TNT005 indicating internalization of these antibodies by cSCC cells (Fig. 4).

Figure 4
figure 4

C1s targeted antibodies TNT003 and TNT005 inhibit ERK1/2 and Akt signaling pathways in cutaneous squamous cell carcinoma (cSCC) cells. A Metastatic cSCC cells (UT-SCC-115) and B non-metastatic (UT-SCC-118) cells were treated with control antibody (IgG) and C1s targeting antibodies TNT003 and TNT005 (250 µg/mL) in serum free conditions and the levels of phosphorylated Akt (p‐Akt), phosphorylated extracellular signal‐related kinase 1/2 (p‐ERK1/2), total Akt, total ERK1/2 and IgG were analyzed in cell lysates by western blotting at time points indicated. Quantitations of the western blots corrected for loading controls are shown below the panels.

Full size image

Discussion

Complement system is an important part of innate immunity. Complement molecules have been shown to play a major role in tumor microenvironment, but also intracellular functions for complement have been discovered10,19. Recent studies emphasize the non-canonical, cascade-unrelated, role of complement in cancer10,19,20,21,22,23. In cSCC, upregulated expression of several complement molecules by tumor cells has been demonstrated7. Tumor cell‒derived complement classical pathway C1 complex components, C1r and C1s and alternative pathway components C3, FB and FD and complement inhibitors FH and FI have been revealed to regulate the progression of cSCC at least partly in non-canonical manner12,13,24,25,26. In complement-targeted therapies the major focus has been in inflammatory disorders and several complement pathway molecule and inhibitor targeted compounds are in clinical trials7. They might offer therapeutic approaches also to cancer.

Previous studies show that C1s is abundantly expressed in tumor cells in invasive cSCCs in vivo, whereas the expression of C1r and C1s is lower in cSCC in situ, actinic keratosis and normal skin12. In this study we took a step forward to investigate the possibility to target human C1s on cSCC cells in culture. We first detected secreted C1s and C1r in cell culture media of cSCC cells, as shown previously12. There is also increasing evidence that complement molecules may function in an intracellular, non-canonical manner. The cleavage of intracellular C3 has been demonstrated in several studies and the biological relevance of the cleavage product C3a in cellular function is undeniable, but the mechanism is still poorly understood27. Intracellular complement inhibitor FH has been detected to promote tumor cell proliferation and migration in cancer cells, such as lung adenocarcinoma28. Additionally, intracellular C1s has been noted to modify the tumor cell phenotype e.g. in clear cell renal cell carcinoma29. Thus, we wanted to study the levels of C1s and C1r in cSCC cell lysates. We noted the expression of C1s in cSCC cell lines and in some cSCC cell lines intracellular levels of C1s were higher than the secreted C1s levels. Additionally, C1r was secreted to the medium or noted as intracellular in the cSCC cell lines that also express C1s. This indicates that C1s may be activated by C1r in these cells, although no correlation between C1r and C1s protein levels produced by cSCC cells has been noted in previous study12. Our data indicate that C1s might also function intracellularly by non-canonical manner in cSCC cells in culture. The fact that no C1q nor downstream complement classical pathway components C2 or C4 are present in cell culture conditions also emphasizes a non-canonical function of C1s in cSCC cells12. Interestingly, the C1s targeted antibodies were detected in cell lysates indicating internalization of the antibodies and potential inhibition of intracellular C1s.

There are several therapeutic strategies to target complement system compounds and inhibitors, such as small molecule inhibitors (SMIs), peptides, and monoclonal antibodies. They are at different stages of drug development both in clinical trials and in the preclinical phase7,

Data availability

In response to reasonable requests, noncommercially available materials that Sanofi has the right to provide, will be made available to not-for-profit or academic requesters upon completion of a material transfer agreement. Requests may be made by contacting Dr. Michael Storek at Michael.Storek@Sanofi.com.

Abbreviations

CAD:

Cold agglutinin disease

CIDP:

Chronic inflammatory demyelinating polyneuropathy

cSCC:

Cutaneous squamous cell carcinoma

C1s:

Complement component 1s

C1r:

Complement component 1r

C3:

Complement component 3

FB:

Factor B

FD:

Factor D

FI:

Factor I

FH:

Factor H

SMI:

Small molecule inhibitor

References

  1. Nehal, K. S. & Bichakjian, C. K. Update on keratinocyte carcinomas. N. Engl. J. Med. 379, 363–374 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Knuutila, J. S., Riihilä, P., Kurki, S., Nissinen, L. & Kähäri, V. M. Risk factors and prognosis for metastatic cutaneous squamous cell carcinoma: A cohort study. Acta Derm. Venereol. 100, 100266 (2020).

    Article  Google Scholar 

  3. Nagarajan, P. et al. Keratinocyte carcinomas: Current concepts and future research priorities. Clin. Cancer Res. 25, 2379–2391 (2019).

    Article  PubMed  Google Scholar 

  4. Ratushny, V., Gober, M. D., Hick, R., Ridky, T. W. & Seykora, J. T. From keratinocyte to cancer: the pathogenesis and modeling of cutaneous squamous cell carcinoma. J. Clin. Invest. 122, 464–472 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bohlson, S. S., Garred, P., Kemper, C. & Tenner, A. J. Complement nomenclature deconvoluted. Front. Immunol. 10, 1308 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ricklin, D., Hajishengallis, G., Yang, K. & Lambris, J. D. Complement: a key system for immune surveillance and homeostasis. Nat. Immunol. 11, 785–797 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Riihilä, P. et al. Complement system in cutaneous squamous cell carcinoma. Int. J. Mol. Sci. 20, 3550 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Venkatraman-Girija, U. et al. Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation. Proc. Natl. Acad. Sci. USA 110, 13916–13920 (2013).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  9. Reis, E. S., Mastellos, D. C., Ricklin, D., Mantovani, A. & Lambris, J. D. Complement in cancer: Untangling an intricate relationship. Nat. Rev. Immunol. 18, 5–18 (2018).

    Article  CAS  PubMed  Google Scholar 

  10. Roumenina, L. T., Daugan, M. V., Petitprez, F., Sautès-Fridman, C. & Fridman, W. H. Context-dependent roles of complement in cancer. Nat. Rev. Cancer. 19, 698–715 (2019).

    Article  CAS  PubMed  Google Scholar 

  11. Bulla, R. et al. C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation. Nat. Commun. 7, 10346 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Riihilä, P. et al. Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma. Br. J. Dermatol. 182, 658–670 (2020).

    Article  PubMed  Google Scholar 

  13. Riihilä, P. et al. Complement factor I promotes progression of cutaneous squamous cell carcinoma. J. Invest. Dermatol. 135, 579–588 (2015).

    Article  PubMed  Google Scholar 

  14. Shi, J. et al. TNT003, an inhibitor of the serine protease C1s, prevents complement activation induced by cold agglutinins. Blood 123, 4015–4022 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Simmons, K. T. et al. Anti-C1s humanized monoclonal antibody SAR445088: A classical pathway complement inhibitor specific for the active form of C1s. Clin. Immunol. 251, 109629 (2023).

    Article  CAS  PubMed  Google Scholar 

  16. Farshchian, M., Nissinen, L., Grénman, R. & Kähäri, V. M. Dasatinib promotes apoptosis of cutaneous squamous carcinoma cells by regulating activation of ERK1/2. Exp. Dermatol. 26, 89–92 (2017).

    Article  CAS  PubMed  Google Scholar 

  17. Johansson, N. et al. Expression of collagenase-3 (matrix metalloproteinase-13) in squamous cell carcinomas of the head and neck. Am. J. Pathol. 151, 499–508 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Wahrmann, M. et al. Effect of the anti-C1s humanized antibody TNT009 and its parental mouse variant TNT003 on HLA antibody-induced complement activation—A preclinical in vitro study. Am. J. Transplant. 17, 2300–2311 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hajishengallis, G., Reis, E. S., Mastellos, D. C., Ricklin, D. & Lambris, J. D. Novel mechanisms and functions of complement. Nat. Immunol. 18, 1288–1298 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cho, M. S. et al. Autocrine effects of tumor-derived complement. Cell Rep. 6, 1085–1095 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kourtzelis, I. & Rafail, S. The dual role of complement in cancer and its implication in anti-tumor therapy. Ann. Transl. Med. 4, 265 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Nissinen, L., Farshchian, M., Riihilä, P. & Kähäri, V. M. New perspectives on role of tumor microenvironment in progression of cutaneous squamous cell carcinoma. Cell Tissue Res. 365, 691–702 (2016).

    Article  CAS  PubMed  Google Scholar 

  23. Afshar-Kharghan, V. The role of the complement system in cancer. J. Clin. Invest. 127, 780–789 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Riihilä, P. et al. Complement component C3 and complement factor B promote growth of cutaneous squamous cell carcinoma. Am. J. Pathol. 187, 1186–1197 (2017).

    Article  PubMed  Google Scholar 

  25. Riihilä, P. M. et al. Complement factor H: A biomarker for progression of cutaneous squamous cell carcinoma. J. Invest. Dermatol. 134, 498–506 (2014).

    Article  PubMed  Google Scholar 

  26. Rahmati Nezhad, P. et al. Complement factor D is a novel biomarker and putative therapeutic target in cutaneous squamous cell carcinoma. Cancers (Basel) 14, 305 (2022).

    Article  CAS  PubMed  Google Scholar 

  27. Zarantonello, A., Revel, M., Grunenwald, A. & Roumenina, L. T. C3-dependent effector functions of complement. Immunol. Rev. 313, 120–138 (2023).

    Article  CAS  PubMed  Google Scholar 

  28. Daugan, M. V. et al. Intracellular factor H drives tumor progression independently of the complement cascade. Cancer Immunol. Res. 9, 909–925 (2021).

    Article  CAS  PubMed  Google Scholar 

  29. Daugan, M. V. et al. Complement C1s and C4d as prognostic biomarkers in renal cancer: Emergence of noncanonical functions of C1s. Cancer Immunol. Res. 9, 891–908 (2021).

    Article  CAS  PubMed  Google Scholar 

  30. Ye, J., Yang, P., Yang, Y. & **a, S. Complement C1s as a diagnostic marker and therapeutic target: Progress and propective. Front. Immunol. 13, 1015128 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sharp, J. A., Whitley, P. H., Cunnion, K. M. & Krishna, N. K. Peptide inhibitor of complement c1, a novel suppressor of classical pathway activation: Mechanistic studies and clinical potential. Front. Immunol. 5, 406 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Travins, J. M. et al. Biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s. Bioorg. Med. Chem. Lett. 18, 1603–1606 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Chen, J. J., Schmucker, L. N. & Visco, D. P. Pharmaceutical machine learning: Virtual high-throughput screens identifying promising and economical small molecule inhibitors of complement factor C1s. Biomolecules 8, 24 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Szilágyi, K. et al. Design and selection of novel C1s inhibitors by in silico and in vitro approaches. Molecules 24, 3641 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Kasprick, A. et al. The anti-C1s antibody TNT003 prevents complement activation in the skin induced by bullous pemphigoid autoantibodies. J. Invest. Dermatol. 138, 458–461 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Rumsey, J. W. et al. Classical complement pathway inhibition in a “Human-On-A-Chip” model of autoimmune demyelinating neuropathies. Adv. Ther. (Weinh) 5, 2200030 (2022).

    Article  CAS  PubMed  Google Scholar 

  37. Freire, P. C. et al. Specific inhibition of the classical complement pathway prevents C3 deposition along the dermal-epidermal junction in bullous pemphigoid. J. Invest. Dermatol. 139, 2417–2424 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. D’Sa, S. et al. Safety, tolerability, and activity of the active C1s antibody riliprubart in cold agglutinin disease: A phase 1b study. Blood 143, 713–720 (2024).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Johanna Markola for her technical assistance, Reidar Grénman for cSCC cell lines and Dr. Norbert E. Fusenig (The German Cancer Research Center, Heidelberg, Germany) for HaCaT cells. This research was funded by Finnish Cancer Research Foundation, Jane and Aatos Erkko Foundation, Sigrid Jusélius Foundation, the State Research Funding of the Turku University Hospital (grant 13336) and Cancer Foundation of the Southwest Finland (P.R.).

Author information

Authors and Affiliations

  1. Department of Dermatology and FICAN West Cancer Centre Research Laboratory, University of Turku and Turku University Hospital, Hämeentie 11 TE6, 20520, Turku, Finland

    Liisa Nissinen, Pilvi Riihilä, Kristina Viiklepp & Veli-Matti Kähäri

  2. Sanofi, Cambridge, MA, USA

    Vaishnavi Rajagopal & Michael J. Storek

Authors
  1. Liisa Nissinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pilvi Riihilä
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kristina Viiklepp
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vaishnavi Rajagopal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael J. Storek
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Veli-Matti Kähäri
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conception and design: LN, VMK; Methodology: LN, PR, KV, VR, MJS; Acquisition of data: LN, PR, KV; Analysis and interpretation of data: LN, VMK; Writing, review, and revision of the manuscript: LN, PR, KV, VR, MJS, VMK; Acquisition of funding: VMK.

Corresponding author

Correspondence to Veli-Matti Kähäri.

Ethics declarations

Competing interests

LN, PR, KV and VMK declare no conflict of interest. VR and MJS are current employees of Sanofi and may hold shares and/or stock options in the company.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nissinen, L., Riihilä, P., Viiklepp, K. et al. C1s targeting antibodies inhibit the growth of cutaneous squamous carcinoma cells. Sci Rep 14, 13465 (2024). https://doi.org/10.1038/s41598-024-64088-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41598-024-64088-3

  • Springer Nature Limited

Keywords